Rapid magnetic flow assays

ABSTRACT

Disclosed is an improvement in methods for nucleic acid and immunological bioassays. The methods comprise a step for “sweeping” paramagnetic bead: target molecule complexes so as to capture them with an affinity capture agent on a test pad by moving a magnetic force field from outside to inside the test pad area so as to bring into contact the paramagnetic complexes with the capture agent, while sweeping any unbound paramagnetic material off the test pad by moving the magnetic field from inside to outside the test pad area. Surprisingly, the paramagnetic complexes are rapidly affinity-extracted from the moving magnetic field.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International PCT PatentApplication No. PCT/US2007/006585, filed Mar. 15, 2007, now pending,which claims the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalPatent Application No. 60/782,649, filed Mar. 15, 2006, and U.S.Provisional Patent Application No. 60/844,811, filed Sep. 14, 2006.These applications are incorporated herein by reference in theirentireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Contract No. UO1AI061187, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 660115_(—)455C1_SEQUENCE_LISTING.txt. The textfile is 6 KB, was created on Sep. 3, 2008, and is being submittedelectronically via EFS-Web.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the general fields of molecular biologyand medical science, and more particularly to improved methods fornucleic acid and immunological bioassays.

2. Description of the Related Art

There has been a dramatic transition in clinical laboratory diagnosticassays from the macroscale to the microscale, with specimen volumerequirements decreasing from milliliters to microliters, and continuingreduction of assay times from hours to minutes.

These improvements are due in part to advances in materials andfabrication, to the rapidity of mass and heat transfer at themicroscale, and to increases in detection sensitivity, but alsorepresent a continuing effort at innovation.

Numerous heterogeneous binding assay systems are known in the prior artand need not be reviewed here. Particles, beads and microspheres,impregnated with color or having a higher diffraction index, are widelyused to speed target isolation. The sensitivity of these assays can beimproved with ELISA, with fluorescent dyes, by fluorescence quenching,with QDots as tags, for example, thereby achieving higher sensitivity,smaller test pads and larger arrays. Increasingly, nucleic acid assaytargets have replaced serological testing. Conventional means fornucleic acid amplification have had a dramatic effect on assaysensitivity and robustness.

However, more can be accomplished to improve sensitivity and acceleratedetection of the assay endpoint. Typically the problem is one ofdiffusion kinetics and mass transfer.

The zeta-potential at the shear boundary layer around particles insolution can slow the close approach needed for binding andimmobilization of an assay target. In U.S. Pat. No. 6,720,411, particlecolloids such as gold colloids coated with oligomers are aggregated insuch a way that the color changes with the state of aggregation. Asillustrated in Example 2B of U.S. Pat. No. 6,720,411, the color changesnoted are reported to occur over several hours. The endpoint istemperature and salt sensitive and thus represents Brownian motion aspath length, the counterion layer as a diffusion barrier, and reductionof interfacial tension as a driving potential. Antibodies can bedetected by similar methods, as illustrated in U.S. Pat. No. 6,974,669.However, these detection methods are inherently slow.

Target migration and complexation rates in a solid chromatographicmatrix also limit the sensitivity and velocity of endpoint detection inlateral flow assays. Immunochromatographic tests, commonly referred toas Lateral Flow Assays, have been widely used for qualitative andsemi-quantitative assays relying on visual detection. One advantage isthe wide variety of analytes that can be detected using this type oftest. Consequently, a large industry exists for commercialization ofthis methodology. See, e.g., U.S. Pat. No. 5,120,643, U.S. Pat. No.4,943,522, U.S. Pat. No. 5,770,460, U.S. Pat. No. 5,798,273, U.S. Pat.No. 5,504,013, U.S. Pat. No. 6,399,398, U.S. Pat. No. 5,275,785, U.S.Pat. No. 5,504,013, U.S. Pat. No. 5,602,040, U.S. Pat. No. 5,622,871,U.S. Pat. No. 5,656,503, U.S. Pat. No. 4,855,240, U.S. Pat. No.5,591,645, U.S. Pat. No. 4,956,302, U.S. Pat. No. 5,075,078, and U.S.Pat. No. 6,368,876 and U.S. Pat. No. 648,982. Techniques for lateralflow assays are discussed in TechNotes #303 “Lateral Flow Tests” byBang's Laboratories, Inc. (Fishers, Ind.), which is incorporated hereinin full by reference.

As another example, in U.S. Pat. No. 5,989,813, amplicons are preparedby amplification of target nucleic acid sequences in the presence offorward and reverse primers conjugated with biotin and digoxigenin,respectively, for use in lateral flow assays. The amplicons are bound toparticles with streptavidin and agglutinate in the presence of antibodyto digoxigenin. By lateral flow in a sorbent, bifunctional ampliconcomplexes are detected as trapped aggregates excluded from the fibrousmatrix. Other solids are interferences in the assay. In a second variantof the lateral flow format, the avidin conjugates are wicked into amembrane and migrate until encountering a detection strip coated with acapture agent. Accumulation of dyed particles at the detection strip isdetected. The assays are generally dependent on flow rate in thematerials, particle size and pore dimensions as well as laminar barriersto diffusion.

In Lateral Flow Assays, it is well known that capillary flow rate andadequate contact between the analyte and its corresponding captureantibody immobilized within the membrane are critical to the assaysensitivity. This demands careful membrane selection to optimize dwelltime and flow rates. Contact between capture antibody and target analyteagain involves convective and diffusional barriers to endpointdetection. These and other limitations of lateral flow assays arediscussed in co-assigned US Patent Application 2007/0042427,“Microfluidic Laminar Flow Detection Strip”, herein incorporated in fullby reference.

It is not uncommon that magnetic beads are used to concentratebioanalytes before or during assay (see for example US 2003/0032028).Beads have several advantages over arrays because beads have a higherspecific surface area, move through the liquid sample matrix, and hencehave more encounters per unit time with an assay target than thecorresponding array. Conceptually, use of magnetic microspheres isgenerally regarded as a concentration step, substituting forcentrifugation or filtration.

Magnetic microbeads are also commonly used to position and contactanalytes with reagents or solid substrates, as for example described inU.S. Pat. No. 5,660,990, U.S. Pat. No. 5,707,807, U.S. Pat. No.6,815,160, 2002/0086443, 2002/0192676, 2003/0215825, 2004/0018611,2004/01211364, 2005/0142582, and cumulative related citationsrepresentative of the prior art, all of which are incorporated here infull by reference. These examples show the breadth of the applicationsfor microbeads. In US 2006/0292588, where magnetic control circuitry forbead washing is provided in an assay apparatus, time to assay endpointis again the critical factor (FIG. 1 of US 2006/0292588, showing 5 hr toendpoint).

Magnetic beads have proven remarkably amenable to surface chemistry, andare routinely derivatized as assay reagents. Such chemistries includefunctional groups selected from carboxylate, amine, amide, hydrazide,anhydride, hydroxyl, sulfhydryl, chloromethyl, aldehyde, glycidyl(epoxy), and others. A broad range of applications exists.

In adapting microbeads to a microfluidics assay format, the problem oflaminar convective and diffusional boundaries again must be overcome tooptimize sensitivity and time to endpoint.

Accordingly, there remains a need for a generally applicable improvementin the sensitivity and speed of endpoint detection in nucleic acid andimmunological bioassays.

Co-assigned patents and patent applications relevant to the developmentmethods for nucleic acid and antibody bioassays in a microfluidic assayformat include U.S. Pat. No. 6,743,399 (“Pumpless Microfluidics”), U.S.Pat. No. 6,488,896 (“Microfluidic Analysis Cartridge”), US PatentApplications 2005/0106066 (“Microfluidic Devices for Fluid Manipulationand Analysis”), 2002/0160518 (“Microfluidic Sedimentation”),2003/0124619 (“Microscale Diffusion Immunoassay”), 2003/0175990(“Microfluidic Channel Network Device”), 2005/0013732 (“Method andsystem for Microfluidic Manipulation, Amplification and Analysis ofFluids, For example, Bacteria Assays and Antiglobulin Testing”), USPatent Application 2007/0042427, “Microfluidic Laminar Flow DetectionStrip”, and unpublished documents “Microfluidic Cell Capture and MixingCircuit”, “Polymer Compositions and Hydrogels”, “Microfluidic Mixing andAnalytical Apparatus,” “System and method for diagnosis of infectiousdiseases”, and “Microscale Diffusion Immunoassay Utilizing MultivalentReactants”, all of which are hereby incorporated in full by reference.

BRIEF SUMMARY OF THE INVENTION

Surprisingly, ligand-tagged paramagnetic microbeads are readilyextracted from a moving magnetic field by formation of molecular tetherswith solid phase substrates coated with affinity ligand-bindingmolecules.

At odds with this finding, the prior art has taught that such moleculartethers are easily broken and that stationary magnetic fields are neededto keep paramagnetic beads immobilized during washing and separation ofbound and unbound beads. Relevant to affinity capture, US 2004/0226348,hereby incorporated in full by reference, states for example, withrespect to paramagnetic microbeads, “A major concern with the bead assayis the amount of force that a few covalent bonds has to hold a bead tothe detection surface” (para 0007), indicating that the strength of acovalent bond is relatively weak. The disclosure continues,“Electromagnets can be controlled to exert a precise amount of force.This is critical in the stage of washing in an assay, where beadsattached to a bottom testing surface are separated from beads that areunattached. During this stage, the precision in the amount of forceapplied to the beads is critical because the difference in force betweenmoving an unattached bead and one that is tethered (i.e., attached) witha few covalent bonds (or biotin/avidin or DNA hybridization) may beextremely slight. Care must be taken to ensure that unattached beads arethe only ones moved and the tethered beads remain attached to anintended surface” (para 0033). It was taught that, “The use ofelectromagnets eliminates the need to design precise flow mechanisms tokeep beads in place” (para 0009). Similar teachings are reported in US2004/0005718, where is stated, “Since magnetic beads to which probe DNAis attached are fixed to the substrate by being attracted by a magneticforce, the magnetic beads can be fixed to the substrate by a strongerforce than the conventional bonding of probe DNA with the substrate”(para 0037), again teaching that a magnetic force is stronger than amolecular binding force.

It was thus unexpected that tagged paramagnetic beads can be affinityextracted from a moving magnetic field, not simply directed to orretained on a test pad by a stationary magnetic field. We found that adetectable endpoint for a bioassay can thus be achieved in one simplestep wherein first a population of ligand-tagged paramagnetic microbeadsis captured on an affinity-binding test pad as a magnetic field movesthe bead complexes across the test pad, and second, as the magneticfield moves away, affinity tagged paramagnetic beads remain bound, butunbound paramagnetic beads are separated and pulled away to waste.

In the preferred method, the magnetic force field has both aperpendicular force vector and a lateral force vector. The paramagneticbeads are attracted to a surface or substrate by a magnetic force fieldemanating from the opposite side of the surface, and as the magneticfield moves laterally, the paramagnetic beads are dragged across thetest pad while following the magnetic flux laterally. Tagged magneticbeads so readily adhere to the test pad in this way that visualdetection endpoints may be used. Although a visual endpoint ispreferable for its simplicity, the invention is not to be construed aslimited to such.

We also show how this improvement in rapidity of the detection step canbe integrated into various classes of assays for nucleic acids, directand indirect assays for immunoactive targets, and other bioassays.Current detection time from sample introduction to detection is about 5min, including filtration, extraction, and amplification.

While microfluidic devices are used in the embodiments of the examplesreduced to practice herein, the invention again should not be construedas limited to such.

The method comprises the steps of:

a) Immobilizing an affinity capture agent within an area on a substratewithin a fluid path, said fluid path with axis of flow, thereby forminga test pad area;

b) Binding a bioassay target molecule to a paramagnetic microbeadreagent in a fluid and contacting the fluid with the substrate withinsaid fluid path;

c) Sweeping the paramagnetic microbead reagent in the fluid into closecontact with the affinity capture agent by moving a magnetic force fieldon a plane parallel to the axis of flow from outside to inside the testpad area, and thereby affinity capturing any bioassay target moleculebound to the paramagnetic bead reagent from said magnetic force field inthe form of a molecular detection complex; and upon forming saidmolecular detection complex, then sweeping from the test pad area anyparamagnetic microbead reagent in the fluid not formed as moleculardetection complex by moving the magnetic force field on a plane parallelto the axis of flow from inside to outside the test pad area; and,

d) Detecting said molecular detection complex in the test pad area.

In another aspect of the invention, we also show thatpeptidyl-conjugates to the 5′ tail of amplification primer sets aregenerally applicable in polymerase-dependent amplification protocols andare further robust, surprisingly retaining full antigenicity and bindingintegrity following amplification. We show that an immobilized antibody,for example a monoclonal antibody, specific to a peptide-conjugatedamplication primer will capture the products of amplification taggedwith the primer. By using a second primer tagged with a second affinityligand, rapid methods for forming target specific detection complexesare readily designed. Peptidyl-conjugated oligonucleotides have notpreviously been used as primers in PCR amplification, or in otheramplification protocols, or used as means for tagging and discriminatingmixed PCR products in multiplex target detection protocols. Thesedetection complexes thus serve essentially as means for interrogating apeptidyl-primer amplicon library. Unexpectedly, this method has morebreadth than prior art methods of tagging primers, which are limited toa few species of binding pairs, permitting simultaneous separation anddetection of an essentially infinite number of amplicons by the step oftagging each amplicon with a unique peptide hapten (herein “peptidylhapten”) and employing the corresponding antibody to capture andimmobilize it. The magnetic bead assay methods illustrated here are oneembodiment of this discovery.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1-4 depict affinity-immobilized paramagnetic target moleculebinding complexes as detection complexes. Shown are four “sandwich”detection complexes involving a paramagnetic target molecule bindingcomplex and a test pad.

FIG. 5 is a pictograph describing (in panel 5A) the use of a vectoredmoving magnetic field to sweep paramagnetic two-tailed ampliconcomplexes across and over two test pads while magnetically contactingthem with two species of capture antibodies immobilized on the testpads, and (in panel 5B) the resultant immuno-immobilized complexes onthe test pad bearing an antibody specific to the paramagnetic targetmolecule complexes.

FIG. 6 is a pictograph describing (in panel 6A) the use of a vectoredmoving magnetic field to sweep paramagnetic antigen:antibody complexesacross and over a test pad while magnetically contacting them withcapture antigen immobilized on the test pad, and (in panel 6B) theresultant immuno-immobilized complexes on the test pad.

FIG. 7 is a pictograph describing (in panel 7A) the use of a vectoredmoving magnetic field to sweep paramagnetic target antibody:antigencomplexes across and over a test pad while magnetically contacting themwith capture anti-antibodies immobilized on the test pad, and (in panel7B) the resultant immuno-immobilized complexes on the test pad.

FIG. 8 is a pictograph describing (in panel 8A) the use of a vectoredmoving magnetic field to sweep paramagnetic target antibody:targetantigen complexes across and over a test pad while magneticallycontacting them with capture antibodies immobilized on the test pad, and(in panel 8B) the resultant immuno-immobilized complexes on the testpad.

FIG. 9 is a sketch showing the use of the method in a microfluidicdetection chamber for a multiplex assay. The magnetic field can be usedto sweep the paramagnetic target molecule complexes across and overmultiple test pads, or to scrub the test pads back and forth with thecomplexes in order to form affinity-immobilized complexes.

FIG. 10 is a conceptual schematic of test pads and test pad arrays asmay be useful in the method.

FIG. 11 is a flow chart depicting steps of a method for detection ofaffinity-immobilized amplicons.

FIG. 12 is a flow chart depicting steps of a method for detection ofaffinity-immobilized target antibody:antibody complexes with antigen.

FIG. 13 is a flow chart depicting steps of a method for detection ofaffinity-immobilized target antibody:antigen complexes withanti-antibody.

FIG. 14 is a flow chart depicting steps of a method for detection ofaffinity-immobilized target antigen:antibody complexes withcomplementary antibody.

FIG. 15 is a reproduction of a photograph of parallel detection chambersin a microfluidic card treated by the inventive method.

FIG. 16 pictographically depicts an affinity-immobilized moleculardetection complex with complex paramagnetic microbead tethered to asolid phase by a two-tailed amplicon complex.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided as an aid in interpreting theclaims and specification herein. Where works are cited by reference, anddefinitions contained therein are inconsistent in part or in whole withthose supplied here, the definition used therein may supplement butshall not supersede or amend the definition provided herein.

1. DEFINITIONS

Test samples: Representative biosamples include, for example: blood,serum, plasma, buffy coat, saliva, wound exudates, pus, lung and otherrespiratory aspirates, nasal aspirates and washes, sinus drainage,bronchial lavage fluids, sputum, medial and inner ear aspirates, cystaspirates, cerebral spinal fluid, stool, diarrhoeal fluid, urine, tears,mammary secretions, ovarian contents, ascites fluid, mucous, gastricfluid, gastrointestinal contents, urethral discharge, synovial fluid,peritoneal fluid, meconium, vaginal fluid or discharge, amniotic fluid,semen, penile discharge, or the like may be tested. Assay from swabs orlavages representative of mucosal secretions and epithelia areacceptable, for example mucosal swabs of the throat, tonsils, gingival,nasal passages, vagina, urethra, rectum, lower colon, and eyes, as arehomogenates, lysates and digests of tissue specimens of all sorts.Mammalian cells are acceptable samples. Besides physiological fluids,samples of water, industrial discharges, food products, milk, airfiltrates, and so forth are also test specimens. In some embodiments,test samples are placed directly in the device; in other embodiments,pre-analytical processing is contemplated.

Bioassay Target Molecule: or “analyte of interest”, or “targetmolecule”, may include a nucleic acid, a protein, an antigen, anantibody, a carbohydrate, a cell component, a lipid, a receptor ligand,a small molecule such as a drug, and so forth. Target nucleic acidsinclude genes, portions of genes, regulatory sequences of genes, mRNAs,rRNAs, tRNAs, siRNAs, cDNA and may be single stranded, double strandedor triple stranded. Some nucleic acid targets have polymorphisms,deletions and alternate splice sequences. Multiple target domains mayexist in a single molecule, for example an immunogen may includemultiple antigenic determinants. An antibody includes variable regions,constant regions, and the Fc region, which is of value in immobilizingantibodies.

Pathogen: an organism associated with an infection or infectiousdisease.

Pathogenic condition: a condition of a mammalian host characterized bythe absence of health, i.e., a disease, infirmity, morbidity, or agenetic trait associated with potential morbidity.

“Target nucleic acid sequence” or “template”: As used herein, the term“target” refers to a nucleic acid sequence in a biosample that is to beamplified in the assay by a polymerase and detected. The “target”molecule can be present as a “spike” or as an uncharacterized analyte ina sample, and may consist of DNA, cDNA, gDNA, RNA, mRNA, rRNA, or miRNA,either synthetic or native to an organism. The “organism” is not limitedto a mammal. The target nucleic acid sequence is a template forsynthesis of a complementary sequence during amplification. Genomictarget sequences are denoted by a listing of the order of the bases,listed by convention from 5′ end to 3′ end.

Reporter, “Label” or “Tag” refers to a biomolecule or modification of abiomolecule that can be detected by physical, chemical, electromagneticand other related analytical techniques. Examples of detectablereporters include, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,dyed particles, spin labels, molecules that emit chemiluminescence,electrochemically active molecules, enzymes, cofactors, enzymes linkedto nucleic acid probes, and enzyme substrates. Reporters are used inbioassays as reagents, and are often covalently attached to anothermolecule, adsorbed on a solid phase, or bound by specific affinitybinding.

Ligand: any molecule for which there exists another molecule (i.e., an“antiligand” or ligand binding molecule) that binds with specificaffinity to the ligand with stereochemical recognition or “fit” of someportion of the ligand by the ligand binding molecule. Forces betweenligand and binding molecule are typically Van der Waals, hydrogen bond,hydrophobic bond, and electrostatic bond. Ligand binding is nottypically covalent and is thus distinguished from “crosslinked” and“derivatized”. As used herein, the term “ligand” is reserved for bindingmoieties that are not “Peptidyl haptens”.

Peptidyl hapten: Refers to a subclass of haptens that is a peptidefragment. As used herein, peptidyl haptens, or “peptide haptens” areused with their complementary antibody to the peptide fragment as ameans for capturing two-tailed amplicons on a solid phase.

Haptens are “molecular keys” in the Kekulean sense, that when bound toan immunogenic carrier and introduced into a vertebrate, will elicitformation of antibodies specific for the hapten or epitope. Thesemolecular keys have stereochemical specificity, are generally exposed onthe surface of the carrier, and are of lower molecular weight than thecarrier. Illustrative examples include small-molecule derivatives ofnative proteins, RNA loop-stem structures, a drug or steroid such asdigoxigenin, the carbohydrate side-chains that decorate a mucopeptide,and short chain peptides or helices of non-native proteins such asdiphtheria toxin or toxoid. Even a dipeptide or a lipid, when conjugatedon a suitable immunogenic carrier, can produce an antibody response, andaffinity-captured antibody specific to the dipeptide or lipid itself,not the immunogen, can be produced by absorbing out the non-specificantibodies in an antiserum or by preparing a monoclonal antibody bylymphocyte selection. Although a hapten is not immunogenic of itself, ithas very finely directed immunospecificity and is recognized by a verylimited set of complementary antibodies.

As used herein, short chain peptides are a preferred hapten for taggingamplicons as used to create peptidyl-amplicon libraries because of theirrobust chemistry, compatibility with enzymes as primer labels, andessentially infinite immunospecificity.

Capture agent: or “affinity capture agent” is a generic term for acomplementary partner in an affinity binding pair and is generally usedto capture a ligand or hapten by binding it to a solid phase. Affinitybinding pairs include streptavidin:biotin, antibody:antigen,hapten:antibody, peptidyl hapten:antibody, and antigen:antibody, forexample, and either member of the affinity binding pair may be thecapture agent.

Test pad area—or test strip, or test field, or simply “test pad”, asused herein, is an area or zone occupied by an affinity capture agent.The area is 3-dimensional at a nanomolecular level and is generallyformed on the surface of a substrate in a liquid flow path. The test padis generally the site in the assay where the assay endpoint is observedor measured, and as such may be housed in a detection chamber withoptical window.

Heterogeneous capture or immobilization: refers use of affinity bindingpairs to concentrate an analyte or detection complex on a solid phasesurface, particle, or porous adsorbent material, generally so that theanalyte can be detected, concentrated or purified. Heterogeneous orsolid phase capture may be achieved with capture agents such asimmobilized antigen, antibody, avidin, nickel-NTA, lectin, or otherligand/receptor systems. As referred to herein, the molecular complexformed by heterogeneous capture is the “immobilized reporter complex”and may be the detection complex of a heterogeneous binding assay. Suchcomplexes are stabilized by non-covalent and cooperative binding.

Amplification: As used here, the term “amplification” refers to a“template-dependent process” that results in an increase in theconcentration of a nucleic acid sequence relative to its initialconcentration. A “template-dependent process” is a process that involves“template-dependent extension” of a “primer” molecule. A “primer”molecule refers to a sequence of a nucleic acid that is complementary toa known portion of the target sequence. A “template dependent extension”refers to nucleic acid synthesis of RNA or DNA wherein the sequence ofthe newly synthesized strand of nucleic acid is dictated by the rules ofcomplementary base pairing of the target nucleic acid and the primers.

Amplicon refers to a double stranded DNA product of a prior artamplification means, and includes double stranded DNA products formedfrom DNA and RNA templates.

Two-tailed Amplicon refers to a double stranded DNA product of a priorart amplification means in which tagged primer pairs are covalentlyincorporated, a first primer conjugated with one affinity tag, a secondprimer conjugated with a second affinity tag, the two tags beingdifferent. As used herein, the two-tailed amplicon functions as a“hetero-bifunctional” tether, and links a magnetic bead to a solidsubstrate.

Primer: as used herein, is a single-stranded polynucleotide orpolynucleotide conjugate capable of acting as a point of initiation fortemplate-directed DNA synthesis in the presence of a suitable polymeraseand cofactors. Primers are generally at least 7 nucleotides long and,more typically range from 10 to 30 nucleotides in length, or longer. Theterm “primer pair” refers to a set of primers including a 5′ “forward”or “upstream” primer that hybridizes with the complement of the 5′ endof the DNA template to be amplified and a 3′ “reverse” or “downstream”primer that hybridizes with the 3′ end of the sequence to be amplified.Note that both primers have 5′ and 3′ ends and that primer extensionalways occurs in the direction of 5′ to 3′. Therefore, chemicalconjugation at or near the 5′ end does not block primer extension by asuitable polymerase. Primers may be referred to as “first primer” and“second primer”, indicating a primer pair in which the identity of the“forward” and “reverse” primers is interchangeable. Additional primersmay be used in nested amplification.

In the preferred embodiment, the first primer is a monospecific orclass-specific oligonucleotide conjugated to a peptide hapten or epitoperecognized by a specific antibody. And the second “primer” is anoligonucleotide conjugated to a hapten, to a biotin, a digoxin, asteroid, a polysaccharide, an antigen or fragment thereof, a folic acid,a phycoerythrin dye, a fluorophore, to an Fc fragment of an antibody, toa nickel chelator such as NTA, or to a lectin, 2,4-dinitrophenyl, and soforth, at or near the 5′ terminus.

Complementary (with respect to nucleic acids) refers to twosingle-stranded nucleic acid sequences that can hybridize to form adouble helix. The matching of base pairs in the double helix of twocomplementary strands is not necessarily absolute. Selectivity ofhybridization is a function of temperature of annealing, saltconcentration, and solvent, and will generally occur under lowstringency when there is as little as 55% identity over a stretch of atleast 14-25 nucleotides. Stringency can be increased by methods wellknown in the art. See M. Kanehisa, Nucleic Acids Res. 12:203 (1984).Regarding hybridization of primers, a primer that is “perfectlycomplementary” has a sequence fully complementary across the entirelength of the primer and has no mismatches. A “mismatch” refers to asite at which the base in the primer and the base in the target nucleicacid with which it is aligned are not complementary.

Complementary (with respect to immunobinding) refers toantibody:immunogen or antibody:hapten binding that is immunospecific.

Magnetic Microbead: refers to a “nanoparticle”, “bead”, or“microsphere”, or by other terms as known in the art, having at leastone dimension, such as apparent diameter or circumference, in the micronor nanometer range. An upper range of such dimensions is 600 um, buttypically apparent diameter is under 200 nm, and may be 1-50 um or 5-20nm, while not limited to such. Such particles may be composed of,contain cores of, or contain granular domains of, a paramagnetic orsuperparamagnetic material, such as the Fe₂O₃ and Fe₃O₄ (α-Fe crystaltype),α′-FeCo, ε-Cobalt, CoPt, CrPt₃, SmCo₅, Nickel and nickel alloys,Cu₂MnAl, α-FeZr, Nd₂Fe₁₄B, NoTi, for example. Preferred are theFerrites, defined as ferrimagnetic or ceramic compound materialsconsisting of various mixtures of iron oxides such as Hematite (Fe₂O₃)or Magnetite (Fe₃O₄) and iron oxides in alloys with other metals. Thesematerials as used generally are particles having dimensions smaller thana magnetic domain, and may be formed into particles, beads ormicrospheres with binders such as latex polymers (generically), silica,as is generally well known and inclusive of such materials as arecommercially available.

Particularly preferred are nanoparticles of Fe₃O₄ with diameters in the50 nm-100 um range as are commercially available for magneticbioseparations. These particles are “superparamagnetic”, meaning thatthey are attracted to a magnetic field but retain no residual magnetismafter the field is removed. Therefore, suspended superparamagneticparticles tagged to the biomaterial of interest can be removed from amatrix using a magnetic field, but they do not agglomerate (i.e., theystay suspended) after removal of the field. Also of interest are nickeland cobalt microbeads. These beads may be reactive with peptidescontaining histidine.

Paramagnetic beads have the property that they align themselves alongmagnetic flux lines and are attracted from areas of lower magnetic fluxdensity to areas of higher magnetic flux density.

It should be recognized that magnetic microbeads may be compositematerials. Such beads may further contain other micro- or nanoparticlesagglomerated with a binder. Composites with RF-tags, QDots,up-converting fluorophores, colloid sols and clays, and the like arecontemplated for use in the present invention. A magnetic bead need notbe formed entirely of a magnetic material, but may instead comprise bothmagnetic and non-magnetic materials.

Microbeads may themselves be colloidal and have chromogenic properties,or may be combined with other colloidal metal particles with chromogenicproperties. Mixed suspensions of differently modified microbeads may beused.

Microbeads are by no means simply commodities. They may be modified withsurface active agents such as detergents to control their rheologicalproperties, as in ferrofluids. The surface of microbeads may be modifiedby adsorption or covalent attachment of bioactive molecules, includingimmunoaffinity agents, antibodies, enzymes, dyes, fluorescent dyes,fluorescent quenchers, oligomers, peptide nucleomers, and the like, andmore specifically by coating with streptavidin or single stranded DNAoligomers, for example. These and other cumulative prior art skills areincorporated herein in full without full recitation of their scope, as afull recitation is unnecessary to understand the principles of thecurrent invention except insofar as to recognize that the microbeads ofinterest herein are comprised of at least one paramagnetic elementtherein, as would be readily recognized by those skilled in the priorarts.

Suitable matrices for microbeads include polystyrene, divinylbenzene,polyvinyltoluene, polyester, polyurethane, with optional functionalgroups selected from SO3, COOH, NH2, Glycidyl (COC), OH, Cl, Tosyl,aldehyde, and sulfhydryl. Particles often range from 0.3 to 5 um orlarger. Latex particles of 100 nm, and 1, 5, 20, 50 or 100 um arecommercially available in bulk. Silica may be used as a matrix or as acapsule. Derivatized silane includes OH, NH2, COOH and more. Particlesoften range from 0.5 to 3 um. Dextran may also be used as a matrix.Particles often range from 20-50 nm. Polysaccharide may also be usedwith silane as silica fortified microbeads of particle size around 250nm. Agarose and cellulose matrices include particles in the range of1-10 um, and may be activated for introduction of functional groups.Protein particles, such as of gelatin and albumin, have long been usedfor magnetic microspheres. These are readily activated for amine,carboxyl, hydroxyl and sulfhydryl linkages with ligands or tags.Liposomes are somewhat more refractory to chemical derivatization, buthave been used to make magnetic particles. Naked iron oxide, and otherparamagnetic metal particles are also known, and may be derivatized byadding sulfhydryl groups or chelators. These particles often have sizesof 5 to 300 nm. Certain types of particle populations are known to beuniform in size; in others the heterogeneity may be controlled orselected.

Such microbeads may be readily prepared. For example, carboxyl-modifiedmicrobeads containing ˜20-60% magnetite are made by dispersing a(magnetite)/styrene/divinylbenzene ferrofluid mixture in water, andemulsion-polymerizing the monomers to trap the magnetite in a polymermatrix of microbeads of ˜1 μm diameter. The magnetite is thus dispersedthroughout the solid beads. Other prior art means for synthesizing andmodifying microbeads are commonly known.

Suitable microbeads for practicing the present invention may also bepurchased from vendors such as Bang's Laboratories, Inc. (Fishers Ind.)and Polysciences, Inc (Warrington Pa.), as well as numerous suppliers ofspecialty modified microbeads such as Bioscience Beads (West WarwickR.I.). Tradenames of such beads, again not as a comprehensiverecitation, include Estapor® SuperParaMagnetic Microspheres, COMPEL™Uniform Magnetic Microspheres, Dynabeads® V MyOne™ Microspheres, and thelike. Cobalt paramagnetic microbeads are sold as Dynabead's MyOne TALON.BioMag Plus microbeads from Polysciences have an irregular shape, andthus more surface area for affinity chemistry.

Populations—of microbeads are generally used to assay populations ofassay targets. A population as used herein refers to a set of memberssharing some common element or property. For example, a population ofbeads may be similar in that the beads share a common tag, such as anavidin coat, or a barcode. A population of nucleic acids comprising anassay target may simply share a target nucleic acid sequence, or maycontain a common tag. A population of antibodies may share a commonspecificity. And so forth.

Paramagnetic and Superparamagnetic are taken as functionally synonymousfor the present purposes. These materials when fabricated as microbeads,have the property of responding to an external magnetic field whenpresent, but dissipating any residual magnetism immediately upon releaseof the external magnetic field, and are thus easily resuspended andremain monodisperse, but when placed in proximity to a magnetic field,clump tightly, the process being fully reversible by simply removing themagnetic field.

Magnetic Force Field: is the volume defined by the magnetic flux linesbetween two poles of a magnet or two faces of a coil. Electromagnets anddriving circuitry can be used to generate magnetic fields and localizedmagnetic fields. Permanent magnets may also be used. Preferred permanentmagnetic materials include NdFeB (Neodymium-Iron-Boron Nd₂Fe₁₄B),Ferrite (Strontium or Barium Ferrite), AlNiCo (Aluminum-Nickel-Cobalt),and SmCo (Samarium Cobalt). The magnetic forces within a magnetic forcefield follow the lines of magnetic flux. Magnetic forces are strongestwhere magnetic flux is most dense. Magnetic force fields penetrate mostsolids and liquids. A moving magnetic force field has two vectors: onein the direction of travel of the field and the other in the directionof the lines of magnetic flux.

Localized Magnetic Field: As used herein, a localized magnetic field isa magnetic field that substantially exists in the volume between thepoles of two magnets, and may be attractive or repulsive.

Robustness: refers to the relative tolerance of an assay format tovariability in execution, to materials substitutions, and tointerferences, over a range of assay conditions. Robustness generallyincreases with the strength of the detection signal generated by apositive result. Robustness negatively correlates with the difficultyand complexity of the assay.

Specificity: Refers to the ability of an assay to reliably differentiatea true positive signal of the target biomarker from any background,erroneous or interfering signals.

Sensitivity: Refers to the lower limit of detection of an assay where anegative can no longer be reliably distinguished from a positive.

Assay endpoint: “Endpoint” or “datapoint” is used here as shorthand fora “result” from either qualitative or quantitative assays, and may referto both stable endpoints where a constant plateau or level of reactantis attained, and to rate reactions, where the rate of appearance ordisappearance of a reactant or product as a function of time (i.e., theslope) is the datapoint. Detection of a “molecular detection complex”,also termed an “immobilized reporter complex”, may constitute an assayendpoint.

Microfluidic cartridge: a “device”, “card”, or “chip” with fluidicstructures and internal channels having microfluidic dimensions. Thesefluidic structures may include chambers, valves, vents, vias, pumps,inlets, nipples, and detection means, for example. Generally,microfluidic channels are fluid passages having at least one internalcross-sectional dimension that is less than about 500 μm and typicallybetween about 0.1 μm and about 500 μm, but we extend the upper limit ofthe range to 600 um because the macroscopic character of the beadsuspensions used here have a dramatic effect on the microfluidic flowregime, particularly as it relates to restrictions in the fluid path.Therefore, as defined herein, microfluidic channels are fluid passageshaving at least one internal cross-sectional dimension that is less than600 um. The microfluidic flow regime is characterized by Poiseuille or“laminar” flow. The particle volume fraction (φ) and ratio of channeldiameter to particle diameter (D/d) has a measurable effect on flowcharacteristics. (See for example, Staben M E et al. 2005. Particletransport in Poiseuille flow in narrow channels. Intl J Multiphase Flow31:529-47, and references cited therein.)

Microfluidic cartridges may be fabricated from various materials usingtechniques such as laser stenciling, embossing, stamping, injectionmolding, masking, etching, and three-dimensional soft lithography.Laminated microfluidic cartridges are further fabricated with adhesiveinterlayers or by thermal adhesiveless bonding techniques, such bypressure treatment of oriented polypropylene. The microarchitecture oflaminated and molded microfluidic cartridges can differ.

Lateral flow Assay: refers to a class of conventional assays whereinparticle aggregation, agglutination or binding is detected by applying aparticle-containing fluid to a fibrous layer such as a permeablemembrane and observing the chromatographic properties as the particlesand particle aggregates move into and through the material. Penetrationof clumps of particles is impeded, whereas free particles penetratebetween the fibers. Similarly, free particles may accumulate as clumpsin zones of the fibrous layer treated with affinity binding agents. Thedevices and methods described here are not lateral flow assays.

“Conventional” is a term designating that which is known in the priorart to which this invention relates.

“About” and “generally” are broadening expressions of inexactitude,describing a condition of being “more or less”, “approximately”, or“almost” in the sense of “just about”, where variation would beinsignificant, obvious, or of equivalent utility or function, andfurther indicating the existence of obvious minor exceptions to a norm,rule or limit.

Herein, where a “means for a function” is described, it should beunderstood that the scope of the invention is not limited to the mode ormodes illustrated in the drawings alone, but also encompasses all meansfor performing the function that are described in this specification,and all other means commonly known in the art at the time of filing. A“prior art means” encompasses all means for performing the function asare known to one skilled in the art at the time of filing, including thecumulative knowledge in the art cited herein by reference to a fewexamples.

Means for extracting: refers to various cited elements of a device, suchas a solid substrate, filter, filter plug, bead bed, frit, or column,for capturing target nucleic acids from a biological sample, andincludes the cumulative knowledge in the art cited herein by referenceto a few examples.

A means for polymerizing, for example, may refer to various species ofmolecular machinery described as polymerases and their cofactors andsubstrates, for example reverse transcriptases and TAQ polymerase, andincludes the cumulative knowledge of enzymology cited herein byreference to a few examples.

Means for Amplifying: Include thermocycling and isothermal means. Thefirst thermocycling technique was the polymerase chain reaction(referred to as PCR) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, Ausubel et al. Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989), and inInnis et al., (“PCR Protocols”, Academic Press, Inc., San Diego Calif.,1990). Polymerase chain reaction methodologies are well known in theart. Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of a targetsequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the template to form reaction products, excess primerswill bind to the template and to the reaction products and the processis repeated. By adding fluorescent intercalating agents, PCR productscan be detected in real time.

One isothermal technique is LAMP (loop-mediated isothermal amplificationof DNA) and is described in Notomi, T. et al. Nucl Acid Res 2000 28:e63.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation(Walker et al. Nucleic Acids Research, 1992:1691-1696). A similarmethod, called Repair Chain Reaction (RCR), involves annealing severalprobes throughout a region targeted for amplification, followed by arepair reaction in which only two of the four bases are present. Theother two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridised to DNA that is present in asample. Upon hybridisation, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Another nucleic acid amplification technique is reverse transcriptionpolymerase chain reaction (RT-PCR). First, complementary DNA (cDNA) ismade from an RNA template, using a reverse transcriptase enzyme, andthen PCR is performed on the resultant cDNA.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPO No. 320 308. In LCR, two complementary probe pairs areprepared, and in the presence of the target sequence, each pair willbind to opposite complementary strands of the target such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR, bound ligated unitsdissociate from the target and then serve as “target sequences” forligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes amethod similar to LCR for binding probe pairs to a target sequence.

Qβ Replicase, may also be used as still another amplification method inthe present invention. In this method, a replicative sequence of RNAthat has a region complementary to that of a target is added to a samplein the presence of an RNA polymerase. The polymerase will copy thereplicative sequence that can then be detected.

Still further amplification methods, described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labellingwith a capture moiety (e.g., biotin) and/or a detector moiety (e.g.,enzyme). In the latter application, an excess of labelled probes areadded to a sample. In the presence of the target sequence, the probebinds and is cleaved catalytically. After cleavage, the target sequenceis released intact to be bound by excess probe. Cleavage of the labelledprobe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989, Proc. Natl. Acad. Sci.U.S.A., 86: 1173; Gingeras et al., PCT Application WO 88/10315). InNASBA, the nucleic acids can be prepared for amplification by standardphenol/chloroform extraction, heat denaturation of a clinical sample,treatment with lysis buffer and minispin columns for isolation of DNAand RNA or guanidinium chloride extraction of RNA. These amplificationtechniques involve annealing a primer which has target specificsequences. Following polymerisation, DNA/RNA hybrids are digested withRNase H while double stranded DNA molecules are heat denatured again. Ineither case the single stranded DNA is made fully double stranded byaddition of second target specific primer, followed by polymerisation.The double-stranded DNA molecules are then multiply transcribed by anRNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, theRNAs are reverse transcribed into single stranded DNA, which is thenconverted to double stranded DNA, and then transcribed once again withan RNA polymerase such as T7 or SP6. The resulting products, whethertruncated or complete, indicate target specific sequences.

Davey et al., EPO No. 329 822 disclose a nucleic acid amplificationprocess involving cyclically synthesising single-stranded RNA (“ssRNA”),ssDNA, and double-stranded DNA (dsDNA), which may be used in accordancewith the present invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H(RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase D, resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al. in PCT Application WO 89/06700 disclose a nucleic acidsequence amplification scheme based on the hybridisation of apromoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Thisscheme is not cyclic, i.e., new templates are not produced from theresultant RNA transcripts. Other amplification methods include “RACE”and “one-sided PCR” (Frohman, M. A., In: “PCR Protocols: A Guide toMethods and Applications”, Academic Press, N.Y., 1990; Ohara et al.,1989, Proc. Natl. Acad. Sci. U.S.A., 86: 5673-567).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu etal., (1989, Genomics 4: 560).

Means for detecting: as used herein, refers to an apparatus fordisplaying an endpoint, i.e., the result of an assay, and may include adetection channel and test pads, and a means for evaluation of adetection endpoint. Detection endpoints are evaluated by an observervisually in a test field, or by a machine equipped with aspectrophotometer, fluorometer, luminometer, photomultiplier tube,photodiode, nephlometer, photon counter, voltmeter, ammeter, pH meter,capacitative sensor, radio-frequency transmitter, magnetoresistometer,or Hall-effect device. Magnetic particles, beads and microspheres havingimpregnated color or having a higher diffraction index may be used tofacilitate visual or machine-enhanced detection of an assay endpoint.Magnifying lenses in the cover plate, optical filters, colored fluidsand labeling may be used to improve detection and interpretation ofassay results. Means for detection of magnetic particles, beads andmicrospheres may also include embedded or coated “labels” or “tags” suchas, but not limited to, dyes such as chromophores and fluorophores, forexample Texas Red; radio frequency tags, plasmon resonance, spintronic,radiolabel, Raman scattering, chemoluminescence, or inductive moment asare known in the prior art. Colloidal particles with unique chromogenicsignatures depending on their self-association are also anticipated toprovide detectable endpoints. QDots, such as CdSe coated with ZnS,decorated on magnetic beads, or amalgamations of QDots and paramagneticFe₃O₄ microparticles, optionally in a sol gel microparticulate matrix orprepared in a reverse emulsion, are a convenient method of improving thesensitivity of an assay of the present invention, thereby permittingsmaller test pads and larger arrays. Fluorescence quenching detectionendpoints are also anticipated. A variety of substrate and productchromophores associated with enzyme-linked immunoassays are also wellknown in the art and provide a means for amplifying a detection signalso as to improve the sensitivity of the assay. Detection systems areoptionally qualitative, quantitative or semi-quantitative. Visualdetection is preferred for its simplicity, however detection means caninvolve visual detection, machine detection, manual detection orautomated detection.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

2. DETAILED DESCRIPTION

Turning now to the figures, we will begin with the products of theprocess—detection complexes—and then describe their method ofproduction. FIGS. 1 through 4 illustrate examples of amplicon capture onimmobilized antibody, target antibody capture on immobilized antigen,target antibody capture on immobilized capture agent, and target antigencapture on immobilized antibody, respectively.

The detection complex of FIG. 1 depicts a paramagnetic bead 1immobilized on test pad 2. The tether between bead and antibody 3 isformed by a two-tailed amplicon 4, in this case with first primer 5tagged with peptidyl hapten 6 and second primer 7 tagged with biotin 8,for illustration. The paramagnetic beads are coated with bound avidin 9.

As shown, the immobilized antibody on the test pad 1 has captured a“two-tailed amplicon” (4), i.e., an amplicon withpeptidyl-oligomer-tagged primer at a first end and biotin-tagged primerat the opposite end. These two-tailed amplicons are synthesized duringan amplification step by providing reagent primer sets in which biotinhas been used to tag a second primer and peptide hapten the first primerby conventional chemistries. In this example, the biotin tagged ampliconis captured by the avidin-coated microbead, and the reporter beadcomplex in turn is then immobilized on the test pad. The two-tailedamplicon thus serves as a heterobifunctional tether. A sufficient numberof immobilized beads, as present in a few microliters of reagent, resultin a distinct visual coloration of the test pad. Biotin is only one suchligand useful in constructing these unique molecular detection complexeswith magnetic beads.

Methods for preparation of affinity-modified microbeads are alsocommonly known. As would be obvious to one skilled in the art, compositemagnetic beads can be prepared with materials such as QDots,fluorophores, dyes, enzymes, RFIDs, and so forth, so as to be readilydetectable by alternative detection means when immobilized on therespective test pads. Detection can involve visual detection, machinedetection, manual detection or automated detection. Methods forpreparation of hapten-tagged primers are also readily extracted from theprior art.

Thus a “positive detection complex” results when an amplicon becomestethered to a test pad as shown in FIG. 1. Necessarily, only thoseamplicons bearing both tags are immobilized on any one test pad. Thosetest pads to which magnetic beads are tethered indicate a positiveresult for one specific two-tailed species of hapten-tagged amplicon.Those test pads to which no magnetic beads are tethered indicate anegative result for the respective hapten-tagged amplicon. Because thespecies of hapten are known and are assigned to a particular forward orreverse primer conjugate, the detection event can be interpreted aspositive detection of the particular nucleic acid sequence correspondingto the target nucleic acid sequence under investigation.

In a preferred method, by using peptidyl-haptens (peptide epitopes)attached to the primer nucleotide sequence as a tag, large libraries ofpeptidyl-hapten-tagged amplicons can be prepared by amplification, andinterrogated by the magnetic bead methods described here. Methods usingthe much more limited prior art toolbox of non-peptide ligands ashaptens or binding agents are not so robust.

Clearly, the biotin:avidin affinity binding pair is one of many ligandbinding pairs that might be chosen for affinity binding. Others includenickel:nickel binding complexes, as may be suitable to nickel-bearingmicrobeads. Or digoxin and digoxigenin and complementary antibodies, orthe antibody Fc fragment and Protein A or Protein G. Antibody-coatedmicrobeads may also be used to capture peptidyl hapten-tagged secondprimers (i.e., a unique peptidyl hapten on both primers), and so forth.

In FIG. 2, we see a second detection complex, again taking advantage ofa bifunctional tether. Paramagnetic bead 20 is immobilized on test pad21 coated with antigen. The tether between bead and antigen 22 is formedby an antibody 23. The paramagnetic beads are coated with boundanti-antibody 24. Species specific anti-antibodies are useful for thismethod, as are also Protein A and Protein G.

In FIG. 3, the roles are reversed. Paramagnetic bead 30 is immobilizedon test pad 31 coated with an anti-antibody 32. The tether between bead30 and antibody 32 is again an antibody 33, but the beads are now coatedwith antigen 34. Species specific anti-antibodies are useful for thismethod.

In FIG. 4, antigen is the target molecule of the assay. Paramagneticbead 40 is coupled to test pad 41 coated with antibody 42 specific forthe antigen. The tether between bead 40 and antibody 42 is now anantigen 43. The beads are coated with an antibody 44 specific for theantigen. The antibodies are not necessarily identical, one antibody maybe a hybridoma antibody, the other a polyvalent antibody, and theantibodies may bind to different recognition sites on a macromolecularantigen, or share a common binding site where multiple binding sites arepresent, for example when the antigen is a viral particle.

We now turn to the step in the method whereby the immobilizedparamagnetic complex is produced. Examples of bead:amplicon capture onimmobilized antibody, bead:target antibody capture on immobilizedantigen, bead:target antibody capture on immobilized antibody captureagent, and bead:target antigen capture on immobilized antibody,respectively, will again be discussed.

In FIG. 5, a key is used to describe the elements of the step. In thefirst panel, FIG. 5A, paramagnetic bead:amplicon binding complexes onthe left are in the process of being “swept” or “dragged” onto, throughand across two patches of immobilized antibody illustrating the testpads. It can be appreciated that at a molecular level the test pads arenot two-dimensional layers, but are in fact 3-dimensional surfacescoated with a layer of bound and unstirred water. The cone of themagnetic force field (long arrow) is moving parallel to the plane of thetest pad, but the magnetic flux lines are experienced by paramagneticparticles as being directed downwardly (short arrow). The magnetic forceis thus seen to have two vectors, one directed “downwardly” and theother “laterally” (relative to the plane of the test pad). Paramagneticparticles are attracted to the magnet from which the magnetic forcefield emanates, and the magnet is positioned under the plane of the testpad and is moving from left to right. The “J-hook” at the base of theamplicons (for illustration) represents a peptidyl hapten that isrecognized by one of the test pads, which are coated with differentantibodies (left vs. right test pad).

In the second panel, FIG. 5B, the magnetic force field has moved pastthe test pads, “sweeping” or “dragging” with it unbound paramagneticparticles while—surprisingly—paramagnetic bead complexes bearing theamplicon have been captured and extracted from the magnetic field, andare seen in the panel to be immunoimmobilized on the test pad coatedwith capture antibody specific for the J-hook of the peptidyl hapten.This occurs much in the same way that a cable captures the tailhook of alanding airplane on an aircraft carrier. The bead complexes are draggedthrough the mat of antibody and liquid crystalline water bound to thetest pad. The magnetic force pulls the complexes down into closeapproach and full contact with the antibody mat, contacting antibody andhapten. The magnetic force (which is a weak force) is not strong enoughto rip the hapten from the primer, or even to rip the antibody from thetest pad, but instead releases the bead complex, which remainsimmobilized on the test pad.

Note that the process of removing unbound paramagnetic material from thetest pads after immunocapture could also be accomplished byrepositioning the source of the magnetic field above the test pad.Paramagnetic beads will always move from a field of less dense magneticflux lines to a field of more dense magnetic flux lines. Thus we can saythat capture is accomplished by sweeping the beads from outside toinside the test pad area, and removal of unbound material isaccomplished by sweeping the beads from inside to outside the test padarea, without reference to particular geometries. The magnetic field mayalso serve to remove the unbound material to waste.

Immobilization is specific. In this illustration, the peptide hapten isrecognized only by the complementary antibody of the right test pad, notthe left, and the bead complexes are therefore immobilized only on theright test pad. Detection of the immunoimmobilized bead complexes isthus a positive detection event and indicates here the presence of thetarget amplicon. Detection of the immobilized complexes can be as simpleas a visual estimate of the color of the test pad before and afterbinding, or a comparison with positive and negative control test pads.Paramagnetic beads typically have a distinct color or can be suitablydyed. More complex detection means may also be used.

In FIG. 6, a key is again used to describe the elements of the step. Inthe first panel, FIG. 6A, paramagnetic bead:antibody binding complexeson the left are in the process of being “swept” or “dragged” onto,through and across a patch of immobilized antibody on the test pad. Thebead antibody complexes consist of a mixture of target antibody (blackbody) and nonspecific antibodies (black tips), all of which have beenbound to the beads by an anti-antibody, for example in the case of anassay for antigen-specific IgG immunoglobins in human serum, a mouse orhybridoma anti-human IgG antibody. The test pad is coated with adsorbedantigen specific for the antibody targeted in the assay.

It can again be seen that the magnetic force field (long arrow) ismoving parallel to the plane of the test pad, but is experienced byparamagnetic particles as being directed downwardly (short arrow). Thereare thus two vectors to the magnetic force, the lateral vectorcorresponding to movement of the magnetic field from left to right andthe perpendicular vector corresponding to the magnetic flux lines whichare not shown. Paramagnetic particles are attracted to the magnet fromwhich the magnetic flux lines emanate, and the magnet is positionedbeneath the plane of the test pad and is moving from left to right. Theparamagnetic particles will follow the motion of the magnetic forcefield, and are pulled against the test pad while being dragged from leftto right.

In the second panel, FIG. 6B, the magnetic force field has moved pastthe test pads, “sweeping” or “dragging” with it nonspecific antibody andunbound paramagnetic particles while—surprisingly—paramagnetic beadcomplexes bearing target antibody specific for the antigen have beencaptured and extracted from the magnetic field, and are seen in thepanel to be immunoimmobilized on the test pad.

In FIG. 7A, paramagnetic bead:antibody binding complexes on the left arein the process of being “swept” or “dragged” onto, through and acrossimmobilized antibody on the test pad. The microbeads are coated withantigen complementary or specific for the target antibody of the assay.The test pad is coated with an anti-antibody, for example in the case ofan assay for antigen-specific IgG immunoglobins in human serum, a mouseor hybridoma anti-human IgG antibody.

It can again be seen that the magnetic force field (long arrow) ismoving parallel to the plane of the test pad, but is experienced byparamagnetic particles as having a downward vector (short arrow).Paramagnetic microbeads are attracted to the magnet from which themagnetic force field emanates, and the magnet is positioned beneath theplane of the test pad and is moving from left to right. The paramagneticbeads are thus pulled down on the test pad, in close contact with thecapture agent, while simultaneously transversing the test pad from leftto right.

In the second panel, FIG. 7B, the magnetic force field has moved pastthe test pads, “sweeping” or “dragging” with it unbound paramagneticparticles while—surprisingly—paramagnetic bead complexes bearing targetantibody have been captured and immunoextracted from the magnetic field.

In FIG. 8A, paramagnetic bead:antigen complexes on the left are in theprocess of being “swept” or “dragged” onto, through and acrossimmobilized antibody on the test pad. The beads are coated with antibodycomplementary for the target antigen of the assay. The test pad iscoated with an anti-antigen antibody.

It can again be seen that the magnetic force field (long arrow) ismoving parallel to the plane of the test pad, but is experienced byparamagnetic particles as being directed with a downward vectorcomponent (short arrow). Paramagnetic particles are attracted to themagnet from which the magnetic force field emanates, and the magnet ispositioned beneath the plane of the test pad and is moving from left toright. The paramagnetic particles will follow the motion of the magneticforce field, and are pulled up against the test pad while being draggedfrom left to right.

In the second panel, FIG. 8B, the magnetic force field has moved pastthe test pads, “sweeping” or “dragging” with it unbound paramagneticparticles while—surprisingly—paramagnetic bead complexes bearing targetantigen have been captured and immunoextracted from the magnetic field.

Surprisingly, in the bound layer of water molecules on the test pad, theintermolecular forces of affinity binding are stronger than the magneticforces on the particles. While not limited by theory, the invention is away of solving a critical problem of bioassays, that of facilitating theclose approach of target and target capture agent by dislodging theboundary or unstirred layer of water at the surface of the capturelayer. At the nanoscale of microfluidics, this barrier is a criticalbarrier in affinity binding. Typically this problem has been overcome byextending incubation time or by convective close approach (for exampleas in the wicking effect of lateral flow) followed by diffusion andcapture. Here we show that unbound paramagnetic complexes are firstbrought into contact with a capture surface or substrate under thedirection of a magnetic force field and are then extracted from themagnetic field, while unbound paramagnetic substrates are dragged awayfrom the capture surface or substrate by the continued lateral motion ofthe magnetic field.

The magnetic force field thus has two vectors, one directed “downwardly”(relative to the plane of the capture surface or test pad) and the other“laterally” (again relative to the plane of the capture surface or testpad). The downward vector penetrates the unstirred water layer aroundthe capture molecule, and draws the target molecule into the requiredclose approach or “close encounter” where affinity binding can occur.The lateral vector is through the unstirred water layer, and again drawsthe target molecules into the required close approach to capturemolecules, but further serves to differentiate bound and unboundmaterial. Unbound paramagnetic molecular complexes remain with themoving magnetic field and continue their lateral path. Boundparamagnetic materials are immobilized at the site of capture and arenot dislodged by the continuing lateral vector of the magnetic forcefield.

The magnetic force field is manipulated by moving its source (apermanent magnet or electromagnet) laterally across or through the planeof the test pad, and may be disengaged by withdrawing the magnet orturning off current to the electromagnet).

In FIG. 9, illustrated is a simple device for conducting the method. Itcan be seen that the lateral motion of the magnetic force field isoptionally bidirectional, here shown with a net motion in a fluid pathof detection chamber 60 from upstream 61 (Paramagnetic Complexes In) todownstream (Waste Out). The source of the magnetic field is again“underneath” (or “behind”) the test pad. This draws the target intoclose approach with the binding sites, and facilitates the detectionstep. In the capture step, the path of the magnetic field mustnecessarily contactingly traverse one or more test pads 62 coated withaffinity capture agent 63. In the detection step, a viewing window 64permits detection of the bound complexes after the moving magnetic fieldhas passed, thus simplifying detection. The magnetic field is furtheruseful in directing unbound paramagnetic materials to waste. A multiplexdetection device is shown, having a plurality of test pads.

Note that this approach to assays eliminates the “hook effect”characteristic of some lateral flow assays. The affinity-modifiedparamagnetic beads are reacted with the target molecule before seeingthe capture agent, and when in excess, do not compete with the targetmolecule for binding on the test pad.

To assemble devices of the kind illustrated in FIG. 9, the test pads aretreated to immobilize a capture agent prior to assembly of the device,for example, on polystyrene, by plasma treatment of plastic areasdelimited by a mask, followed by application of the capture agent anddrying. Spotting of capture agent, for example with a laserjet printer,can eliminate the need for masking test pads. If necessary, test padsare “blocked” with blocking agents to prevent non-specific adsorption oftarget molecules prior to sealing the detection chamber or channel.

In FIG. 10 we show examples of possible test pad geometries. Test padsare a feature of the detection step of the method described herein. Testpads 70 and 71 constitute for example a negative and positive detectionfield for an assay and may be used as a pair. Test pad array 72 is avertical stack of banded or striped test pads in the form of an array,not unlike that shown in FIG. 9. Test pad array 73 is a rectangulararray of individual test squares, each treated with a unique captureagent. Test pad 74 is circular and is adapted to inkjet printing. Testpad 75 is treated with a gradient of a capture agent so as to display areadily interpretable semi-quantitative endpoint.

Test pads have in common a test field bounded by an edge inside of whicha bioactive capture agent is immobilized. While not a comprehensivelist, the capture agent may be a protein such as an antibody, ananti-antibody, an anti peptidyl hapten antibody, Protein A, Protein G,or antigen, or a non-protein such as an aptimer, a carbohydrate antigen,a mucopolysaccharide, a binding protein such as folic acid bindingprotein or an avidin, or a nucleotide oligomer. Capture agents may alsoinclude denatured viral antigens and microbial antigens in general andcellular components or whole cells in general.

Note that test pads are not necessarily impermeable substrates, and maybe porous or fibrous in character. The microbead fluid path in themagnetic field may be across or through the test pad area, as in fromside-to-side or from front-to-back. The test pad architecture, at amolecular level, is inherently three-dimensional, although it may berepresented as a two-dimensional plane.

Solid substrates for test pads include olefin or other thermoplasticmaterials such as polystyrene, polycarbonate, polypropylene,polyethylene terephthalate, polyether sulfone, polyvinyl chloride,polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride, andpolyamides and also inorganic materials such as glass. Certain fibrousor porous supports such as nitrocellulose, nylon, hydrogel, andpolyethylene may also be applied as test pads, and may be pretreatedwith capture agent for ease of assembly. To enhance binding of captureagents, crosslinked proteins are sometimes employed. Drying alsopromotes irreversible binding of the capture agent.

A preferred method for pretreating plastic prior to adsorbing thecapture agent is low pressure gas plasma treatment. Exposure of thesurface to pure oxygen or nitrogen produces an activated hydroxylatedand carboxylated substrate layer or an activated aminated andnitroxidated layer, respectively. Argon may also be used. In oneembodiment, polystyrene plastic is used as the substrate forimmobilizing capture agent. Masking, followed by gas plasma treatment isused to activate designated areas as test pads. The capture agent isapplied, dried in place, and the mask is removed. When antibody is usedas the capture agent, application by hand or with an automated printeris followed by drying and blocking. Other capture agents may requiremodified protocols as are known in the art.

Techniques for surface activation are reviewed in Chan et al. (1996)Surface Science Reports 24:1-54 and in Garbassi et al. (1998) PolymerSurfaces-From Physics to Technology (John Wiley pp 238-241), and in U.S.Pat. No. 6,955,738, which describes hydrophilization andfunctionalization of polymer surfaces and is incorporated herein in itsentirety by reference.

We now disclose integrated assay methods relying on a step for laterallymoving magnetic fields to contactingly capture and extract targetanalytes from biological samples.

Turning to FIG. 11, an assay for a target nucleic acid sequence becomesthe steps of first preparing the sample for amplification of the targetsequence, amplifying the target by a PCR or related isothermal protocolwhereby tagged primers are incorporated into the product amplicon, thenbinding those tagged “two-tailed” amplicons on paramagnetic beads coatedwith an affinity binding agent, and magnetically sweeping or draggingthe beads into close contact with a test pad area with immobilizedcapture antibody so as to form immunoimmobilized paramagnetic complexesof test pad:capture antibody:amplicon with hapten tag of first primerand ligand tag of second primer:binding agent and paramagnetic bead(i.e., the detection complex), before sweeping from the test pad anyun-immobilized paramagnetic material. And finally a step for detectingany molecular detection complexes on the test pad.

Preparation of a sample may involve lysing cells to release the targetnucleic acids, removing interferences such as hemoglobin from a bloodlysate by selective adsorption and elution of the nucleic acids from aglass solid phase, and dissolution of the nucleic acids with a suitablebuffer for a polymerase. Also required in some applications arepreliminary steps for reverse transcription, as when mRNA contains thetarget sequences and must be converted to duplex DNA beforeamplification.

In the step for amplification, multiplex or nested primer sets may beused. The method uses a second primer with tag suitable for complexationwith an affinity binding agent on the paramagnetic beads, and often thisa biotin tag as illustrated in FIG. 1. The method uses a first primerwith tag suitable for immunoaffinity immobilization of the formedamplicon:bead complex on the surface of the test pad.

So two levels of affinity capture are involved, the first being thebinding of a ligand-tagged amplicon on the paramagnetic bead, and thesecond the immobilization or capture of an amplicon:bead bindingcomplexes on the test pad (forming the detection complex or immobilizedreporter complex). Various affinity binding agents may be used in eachphase of formation of the detection complex. However, the advantage ofusing capture antibodies for second phase immobilization is thespecificity of antibody:peptidyl hapten binding, which permits design ofprotocols for simultaneous assay of multiple target nucleic acidsequences. Immuno-immobilization of target analyte with antibody captureagent is a preferred embodiment, but the invention is not limited tosuch.

Having formed the paramagnetic bead:amplicon binding complexes in freesolution, the next step is to use a magnetic field to localize andcontact the analyte complexes with the test pad so that theimmunoimmobilized detection complexes can be formed. The magnetic fieldis moved and optionally modulated to perform this. Lateral motion of themagnetic field sweeps or drags the bead complexes onto the test pad,through the unstirred layer and the 3-dimensional network of boundcapture antibody, and finally across the test pad, where unboundparamagnetic material is carried off the test pad and away with thelateral motion of the magnetic force. This step promotes bindinginteractions without the need for multi-minute incubations.

In the detection step, the double-stranded, two-tailed amplicon, boundby avidin:biotin on one end (for example) and antibody:peptidyl haptenon the other (for example), is sufficiently strong to selectively tetherthe paramagnetic bead to the test pad and resist delocalization by themoving magnetic force field. It can be said that the capture antibody“extracts” the amplicon:bead complexes from the moving magnetic field.Sufficient numbers of bound bead complexes are readily identified andform a positive result by visual endpoint. A visual detection step isillustrated.

It should be noted that the primer set is essentially a first assayreagent, and may be prepared and placed in an assay device or kit,optionally in dried form, at any time prior to performing the assay.Similarly, the beads are essentially a second assay reagent, and may besensitized with the desired binding agent, and optionally dried in placeprior to the assay. Test pads are prepared in advance of the assayitself and may be rehydrated prior to use or rehydrated by the testsample in performance of the assay. Drying promotes irreversible bindingof the capture agent to the test pad substrate. Reagents for samplepreparation and amplification may also be prepared separately.

In FIG. 12 we see the same principles illustrated in a target antibodyassay. The sample is first processed to prepare a liquid fraction, whichmight be serum or plasma, a paracellular fluid, saliva, or otherbiological sample. Generally any solid fraction of the sample isseparated from the aqueous liquid fraction.

Optionally, interferences are then adsorbed and any antibody:targetantigen complexes in the biological sample are disrupted so as torelease the analytical target.

The target antibody in free solution is then bound by paramagnetic beadscoated with an anti-antibody. This method is of use, for example, when aparticular class of target antibody is of interest, as in distinguishingacute, convalescent, and chronic stages of infection, or when allantibody in the sample is to be interrogated for specificity to aplurality of antigens.

In the detection step, the bead:antibody:antibody:antigen tether, issufficiently strong to selectively anchor the paramagnetic bead to thetest pad and resist disruption by the magnetic field. Sufficient numbersof bound bead complexes are readily identified and form a visuallypositive detection endpoint. The detection complex is formed of testpad:antigen:target antibody:affinity bound paramagnetic bead.Alternatively, the detection complex may contain an enzyme, for example,and may be further developed for detection by enzymatic assay.

The common step in all these assays is to simultaneously use a magneticfield a) to localize and contact the analyte:bead complexes with thetest pad so that the immobilized detection complex can be formed andfurther b) to separate bound and unbound paramagnetic bead complexes.This speeds the analytical process. The magnetic field is moved andoptionally modulated to perform this. Lateral motion of the magneticfield sweeps or drags the bead complexes onto the test pad, through theunstirred layer and the 3-dimensional network of bound capture antigen,and finally across the test pad, where unbound paramagnetic material iscarried off the test pad and away with the lateral motion of themagnetic force. Paramagnetic bead complexes bearing target antibodyremain behind, immuno-immobilized on complementary, irreversiblyadsorbed antigen on the test pad.

Clearly the bifunctional or “two-tailed” tether confers assayspecificity. Using FIG. 12 for illustration, which is copacetic withFIGS. 2 and 6, nonspecific antibody may be bound to the paramagneticbeads, but would not be captured by the antigen on the test pads, so nofalse-positive detection complex will form. Anti-antibodies directed atthe Fc fragment of the target antibody are preferable for this assay sothat the variable regions of the target antibody arms are free torecognize and bind to the bound antigen.

It should be noted that the beads are essentially a first assay reagent,and may be sensitized with the desired binding agent, and optionallydried in place prior to the assay. Test pads are prepared in advance ofthe assay itself and may be rehydrated prior to use or rehydrated by thetest sample in performance of the assay. Reagents for sample preparationmay also be prepared separately.

In FIG. 13, the method differs from that of FIG. 12 essentially by thepolarity of the tether. In the sample preparation step, generally, thesolid fraction of the sample is separated from the aqueous liquidfraction. If needed, interferences are then adsorbed and anyantibody:target antigen complexes in the biological sample are disruptedso as to release the analytical target.

The target antibody in free solution is then bound by paramagnetic beadscoated with complementary antigen, forming immunospecificantibody:antigen complexes on the bead (also termed a “reporter:analytecomplex”.

The next step is common to all these assays and involves thesimultaneous use a magnetic field to a) localize and contact theanalyte:bead complexes with the test pad so that the immobilizeddetection complex can be formed and b) to separate bound and unboundparamagnetic bead complexes. Lateral motion of the magnetic field sweepsor drags the bead complexes onto the test pad, through the unstirredlayer with a downward vector on the paramagnetic beads, penetrating the3-dimensional network of bound capture anti-antibody on the test pad,and finally across the test pad, whereupon unbound paramagnetic materialis carried away with the lateral motion of the magnetic force.Paramagnetic bead complexes bearing target antibody remain trapped byimmunoimmobilization on adsorbed anti-antibody on the test pad.

In the detection step, the bead:antigen:antibody:antibody tether, issufficiently strong to selectively anchor the paramagnetic bead to thetest pad and resist disruption by the magnetic field. Sufficient numbersof bound bead complexes are readily identified and form a positivevisual detection endpoint. The detection complex is formed of testpad:antibody:target antibody:affinity bound paramagnetic bead. Thedetection endpoint may be further developed to amplify the detectionsensitivity, for example by excitation of a fluorophore.

Clearly the bifunctional tether confers assay specificity. Using FIG. 13for illustration, which corresponds to FIGS. 3 and 7, the captureanti-antibody on the test pad in panel 13B will likely capture a broadspectrum of antibodies in the sample, but only those immunocomplexed byparamagnetic beads will result in a positive assay. Thus thebifunctional specificity of the tether ensures assay specificity. Notethat, however, the paramagnetic bead reagent is a mixture of beadscoated with either a first antigen and beads coated with a secondantigen, an immunoimmobilized positive assay endpoint will form ifantibodies to either antigen are present in the sample. The identity ofindividual antibodies to particular antigens is, however, obtained withthe method of FIG. 12, even in a multiplexed bead format.

It should be noted that the beads are essentially a first assay reagent,and may be sensitized with the desired binding agent, and optionallydried in place prior to the assay. Test pads are prepared in advance ofthe assay itself and may be rehydrated prior to use or rehydrated by thetest sample in performance of the assay. Reagents for sample preparationmay also be prepared separately.

Similarly, individual antigens in a biological test sample may beidentified by the method of FIG. 14. In FIG. 14 we see the sameprinciples illustrated to assay for a target antigen. The sample isfirst processed to prepare a liquid fraction containing a solution orsuspension of the target antigen.

Optionally, interferences are adsorbed and any antibody:target antigencomplexes in the biological sample are disrupted so as to release theanalytical target.

The target antigen in free solution is then bound by paramagnetic beadscoated with an antibody complementary for the antigen. Multiple antigensmay be targeted simultaneously. This method is of use, for example, whena sample is suspected of carrying an enteric pathogen, a virus, or amarker released from malignant cells.

The common step in all these assays is use a magnetic field to a)localize and contact the analyte:bead complexes with the test pad sothat the immobilized detection complex can be formed and to b) separatebound and unbound paramagnetic bead complexes. Essentially this is donesimultaneously, thus speeding the assay and eliminating multi-minuteincubations for the binding interaction.

The step for magnetic sweeping is comprised of applying a magnetic forceto said paramagnetic bead reagent, wherein said magnetic force comprisesgenerally lateral and generally perpendicular force vectors generated bya moving magnetic force field comprising flux lines extending from lessdense to more dense. Because paramagnetic beads move from areas of lessdense magnetic flux to areas of more dense magnetic flux, the magneticforce pulls the beads onto and into the arms of the capture agent.Because the magnetic field is moving laterally, the magnetic forcesweeps or pulls the beads laterally over and across the test pad,separating bound and unbound materials as it goes. Rates of motion(linear velocity) for the magnetic sweep have been in the range of 25 to100 mm/min (up to about 0.2 cm/sec). This step can be performedmanually, or can be performed with an automated or semi-automatedapparatus.

FIG. 15 illustrates a result of the assay. Seven vertically elongatedetection chambers are placed side by side on a microfluidic cartridgeunder an optical window. Within each detection chamber are seven testpads stacked vertically. Each test pad is about 0.5×2 mm in size.Paramagnetic microbead reporter complexes are added to the detectionchamber via a sample port and the beads are drawn up the detectionchamber by a magnetic field originating from a magnet behind thecartridge. This magnetic field serves to a) draw the reporter complexesto the site of immobilization and b) remove unbound material. The resultis a striking rust colored band where reporter complexes are bound tothe corresponding antibody on a test pad. Seven amplicons were used inpreparation of this test cartridge, and seven corresponding antibodytest pads were prepared in each detection chamber. The result thusappears as a “stairstep” from left to right.

We can thus, in general, characterize the method as a rapid bioassayprotocol comprising a step of moving a magnetic force field from outsideto inside a test pad area so as to sweep a paramagnetic bead reagent ina fluid into close contact with an affinity capture agent in said testpad area, and thereby affinity capturing or extracting any bioassaytarget molecule bound to the paramagnetic bead reagent from the magneticforce field in the form of an immobilized paramagnetic microbeadcomplex; and upon forming the immobilized paramagnetic bead complex(i.e., the detection complex), then moving the magnetic force field frominside to outside the test pad area so as to sweep from the test padarea any paramagnetic bead reagent not formed as immobilizedparamagnetic complex, before detecting the detection complex, althoughit should be clear that, simplicity of description aside, the sweepingstep in fact simultaneously integrates multiple simultaneous acts offormation of immobilized bead complexes and parallel acts of separationof not immobilized materials.

Surprisingly, the tether is sufficiently strong to selectively anchorthe paramagnetic bead to the test pad while resisting the separatingforce of the magnetic field. In the detection step, sufficient numbersof bound bead complexes are readily identified and form a visualdetection endpoint. The detection complex comprisesbead:antibody:antigen:antibody:test pad, and may be further developed toincrease assay sensitivity, for example by exciting an RFID tag or afluorophore embedded in the bead matrix. The bead thus acts as areporter group itself, or as a complex with accessory reporter groups.

Clearly the bifunctional or “two-tailed” tether confers assayspecificity. Using FIG. 14 for illustration, which is copacetic withFIGS. 4 and 8, specific antibody binds the target antigen, such as adrug or other small molecule, to the bead, and the target antigen:beadbinding complex is bound to the test pad again by another antibodyspecific to the target analyte. Specificity and robustness is alsodemonstrated in FIG. 15.

It should be noted that the beads are essentially a first assay reagent,and may be sensitized with the desired binding agent, and optionallydried in place prior to the assay. Test pads are prepared in advance ofthe assay itself, are advantageously dried in place, and may berehydrated prior to use or rehydrated by the test sample in performanceof the assay. Reagents for sample preparation may also be preparedseparately before use.

In the various applications noted above, we have developed a preferencefor monosized bead reagents with high density relative to typicalaqueous solutions. Metallic microbeads settle quickly in micron-sizedflow paths and the beads are not readily resuspended during washing.Interestingly, in certain microfluidic applications, a magnet is nolonger used for routine washing and rinsing of magnetic beads. Thesepreferred beads are also readily detected visually. Labelled test padsappear as brightly rust colored spots or bands on a white or clearbackground.

The size of magnetic beads preferred in the assay are about 0.01 to 50microns, more preferably 0.5 to 10 microns, and most preferentially 0.8to 2.8 microns, mean diameter. Homogeneously sized beads are preferred.Suitable beads may be obtained from Dynal Invitrogen (Carlsbad Calif.),Agencourt Bioscience Corp (Beverly Mass.), Bang's Laboratories, Inc.(Fishers Ind.), Polysciences, Inc (Warrington Pa.), Bioscience Beads(West Warwick R.I.), Bruker Daltonics (Nashville Tenn.) and AGOWA(Berlin Del.), for example.

The magnetic beads may be in the form of a ferrofluid, taken broadly. Inoperation, in traversing the test pad, the method serves as a sort ofmagnetic fluidized bed reactor for extraction of affinity captured beadsand separation out of nonspecifically labeled beads, reagents and assaymaterials.

To effect motion of the magnetic force field relative to the test pad,several alternative embodiments are possible: a) The magnet itself canbe moved. Movement can be manual or powered with a stepper motor, servomotor, voice coil or with a spring-loaded mechanism and an x-z or y-zcarriage can be constructed and automated. Alternatively, b) the testpad may be moved relative to the magnetic field by similar means. And ifelectromagnets are used in place of permanent magnets, c) an array ofelectromagnets can be actuated in sequence to redirect the magneticfield. It is possible to build a solid state system where a series ofelectromagnets are used to move the beads in a chamber. However, themethods of the inventions should not be construed as being limited to amicrofluidic device. Adaption to laminar flow, lateral flow, capillary,dipstick, multiwell plate, and test tube formats is also contemplated.

In a preferred apparatus, as built, a stepper motor is used to move arare earth magnet (neodymium) in an undercarriage mounted in closeproximity to the detection chamber of a microfluidic device. Simplesoftware commands are used to move the undercarriage along y-axis of thedetection chamber (see FIG. 9). The speed of translation is adjustableand the carriage may be lowered in the z-axis to weaken the magneticfield in the chamber. Because paramagnetic beads line up on magneticflux lines and are attracted to areas of higher magnetic flux, theycannot be repelled by the magnet and the orientation of the poles of themagnet is reversible.

The preferred apparatus accepts a microfluidic cartridge with detectionchamber or “microchannel” configured in the body, the microchannelcomprising a fluid path with axis of flow and with upper and loweraspects.

Within the microchannel is a test pad or solid phase element, whichcomprises an affinity capture agent for the analyte or for an analytebinding complex. A means is provided for introducing a population ofparamagnetic microbeads in a fluid into the microchannel, generally byassembling the cartridge with dehydrated beads inside and thenrehydrating the beads in test sample fluid so that the beads complextarget analyte. Also provided is a means for moving a magnetic forcefield along a plane parallel to the axis of flow of said fluid path, soas to sweep the population of paramagnetic microbeads in said fluid intoclose contact with said affinity capture agent, thereby affinitycapturing any bioassay target molecule bound to said population ofparamagnetic beads from the magnetic force field in the form of anmolecular detection complex, and sweeping from the solid phase elementany paramagnetic bead reagent not formed as molecular detection complex.

The means for moving a magnetic force field comprises a subassemblyexternal to said microfluidic cartridge, said subassembly with moveablecarriage with track upon which said carriage is mounted, said trackmounted in a plane parallel to said axis of flow, said carriage furthercomprising a first magnet, the subassembly further configured to movethe magnet along said track, first bringing the magnetic force fieldinto proximity to said test pad and then distancing the magnetic forcefield from said test pad element.

Neodynium (NdFeB) magnets obtained from K&J Magnetics (Jamison Pa.) werefound to be suitable. Magnets designated D38, D40, and D44 were used.These magnets are cylindrical with poles on the long axis and have aCurie temperature of about 300° C. (maximum operating temperature of 80°C.). The magnets are Grade N52 neodynium and have a surface fieldstrength of 4600 to 5000 Gauss. It should be recalled that magneticfield force is inversely proportionate to the 4^(th) power of thedistance. Proximity to the test pad is in the range of 0.2 to 1.2 mm forthese particular magnets. The diameter of the magnets range from toabout 5 to 10 mm at the poles. For reference, the test pads themselvesare about 0.5 mm×2 mm, with the long axis perpendicular to the traverseof the magnet.

Magnets with a triangular cross-section (prism magnets) and poles on twofacets may also be used. These magnets have a sharply focused fluxdensity above the apex of the facets.

Another aspect of the invention is use of peptidyl primer taggedamplicons in assays for nucleic acids. A number of methods are nowavailable for manufacture of specific peptide epitopes attached tooligonucleotide probes or primers (see C.-H. Tung and S. Stein,Bioconjugate Chem., 2000, 11, 605-618; E. Vives and B. Lebleu,Tetrahedron Lett., 1997, 38, 1183-1186; R. Eritja, A. Pons, M.Escarcellar, E. Giralt, and F. Albericio, Tetrahedron Lett., 1991, 47,4113-4120; J. P. Bongartz, A. M. Aubertin, P. G. Milhaud, and B. Lebleu,Nucleic. Acids Res., 1994, 22, 4681-4688; C.-H. Tung, M. J. Rudolph, andS. Stein, Bioconjugate Chem., 1991, 2, 461-465; J. G. Harrison and S.Balasubramanian, Nucleic. Acids Res., 1998, 26, 3136-3145; S.Soukchareun, J. Haralambidis, and G. Tregear, Bioconjugate Chem., 1998,9, 466-475; K. Arar, A.-M. Aubertin, A.-C. Roche, M. Monsigny, and M.Mayer, Bioconjugate Chem., 1995, 6, 573-577; and, for an example of theuse of the native ligation technique see: D. A. Stetsenko and M. J.Gait, J Organic Chem., 2000, 65, 4900-4908). See also US 20006/0263816,incorporated herein in full by reference.

The peptidyl hapten conjugated primers of this method are satisfactorilysynthesized by the above chemistries and others. We disclose here thatprimers of this class are compatible with PCR methods and with molecularbiological nucleic acid amplifications in general. For use in assays,the amplification product with peptide-tagged primer-labelled ampliconsis first captured by an affinity capture agent specific for a ligand onthe second primer of the amplification primer set and bound to amagnetic microbead. The amplicon-bead complex is then interacted withpeptidyl hapten-specific antibodies on the testpad and only those beadcomplexes with the peptide :amplicon molecular complex are captured bythe testpad. This method permits screening of peptidyl-ampliconlibraries by heterogeneous binding assays using magnetic beadtechnology.

The method results in an inventive composition as a product: a moleculardetection complex comprising a two-tailed amplicon with first end andsecond end, said first end comprising a first primer covalentlyconjugated with a peptidyl hapten, and said second end comprising asecond primer covalently conjugated with a ligand, said first endfurther comprising a ligand-bound ligand binding agent-coated reportergroup, and said second end further comprising a peptidyl hapten boundanti-peptidyl hapten antibody immobilized on a solid phase.

This aspect of the invention is illustrated in FIG. 16, which shows amolecular detection complex with two-tailed amplicon as tether, as inFIG. 1, but here not involving biotin, and utilizing a more complexmagnetic microbead than that described in FIG. 1.

In FIG. 16, the magnetic microbead 161 contains inclusion bodies orpatches 162 of QDot, Texas Red, phycoerythrin, or other fluor,covalently attached or immobilized in the magnetic microbead matrix,here a latex binder with embedded particles of a ferrofluid. Themicrobead further comprises adsorbed antibody 169. This is the reportergroup. The tether consists of amplicon 164 with first primer 165 andpeptidyl hapten tag 166. Primer conjugates are incorporated into theamplicon during amplification. Anti-peptidyl hapten antibody 166immobilizes the tether to solid substrate 163, which may be another beador a fiber or a test pad. The second primer 167 is conjugated withdigoxigenin (for example). And the antibody in the reporter group isspecific for digoxigenin. The reporter group is thus immobilized in acomplex comprising at least 5 non-covalent bonds—bead:antibody;antibody:ligand; DNA:DNA; peptide:antibody; and antibody:solid phase.Yet the assay method described here permits the tether and reportergroup to be extracted with high specificity and robustness from a movingmagnetic field in which the paramagnetic beads are carried.

It should be noted that soluble reporter groups and fluorophore dyedlatex beads may be used with the two-tailed amplicons of the presentinvention. By barcoding the fluorophore beads and coating uniformlylabeled bead populations with peptidyl hapten specific antibodies, beadlibraries can be synthesized for analysis of mixed populations oftwo-tailed amplicons or of two-tailed amplicon libraries, and theresulting affinity binding complexes with pairs of beads tethered by thetwo-tailed amplicons can then be sorted or assayed using dual excitationfluorometry, a sort of liquid microarray. These assays may be performed,for example, in a microfluidic cartridge configured as a fluorescentparticle sorter, or in a flow luminometer. In a preferred assay method,the reporter group is a fluorophore of one emission frequency and thebarcoded latex bead is selected from those of the prior art.

Assays of the method described herein are generally amenable to thepreparation of devices, apparatuses, and kits for their performance.

EXAMPLE 1 A) Preparation of Primer Sets

Reverse primers were first prepared and HPLC purified. Peptides werederivatized with n-terminal hydrazine before use. Oligonucleotides weretreated with succinimidyl 4-formylbenzoate in formamide and then reactedwith the hydrazine derivatized peptides to form hapten-tagged primers.

The following peptidyl hapten-tagged primers were used.

5′-Peptidyl Oligomers Pri- mer Primer Sequence* Peptide Sequence** ACGCCAGTACGATATTCAG (HNA) EQKLISEEDL (SEQ ID NO:1) (NH2) (SEQ ID NO:8) BACCTGGACATCACGGCTTTCAAC (HNA) YPYDVPDYA (SEQ ID NO:2) (NH2) (SEQ IDNO:9) C CCTATTGCAGAGCGAATGAC (HNA) YTDIEMNRLGK (SEQ ID NO:3) (NH2) (SEQID NO:10) D TGAACTCCATTAACGCCAGA (HNA) CEEEEYMPME (SEQ ID NO:4) (NH2)(SEQ ID NO:11) E CGACCTGACCAAATGCCAG (HNA) TDFYLK (NH2) (SEQ ID NO:5)(SEQ ID NO:12) F CCTATAACAGCACCCACTATACGG (HNA) DTYRYI (NH2) (SEQ IDNO:6) (SEQ ID NO:13) G CTCTGCGAGCATGGTCTGG (HNA) QPELAPEDPED (SEQ IDNO:7) (NH2) (SEQ ID NO:14)

These peptide epitopes were selected based on the availability ofcomplementary antibodies. Alternate peptide conjugation chemistries mayalso be used. Forward primers were all conjugated with biotin.

B) Preparation of Paramagnetic Microbeads

Monodisperse streptavidin-coated magnetic beads (MyOne Streptavidin ClDynabeads) were purchased from Dynal, Carlsbad Calif. and washed andresuspended in 0.9×PBS, 30 mg/mL BSA and 1% TritonX100 with 5% (v/v) ofa solution of 80 mM MgCl₂, 0.24% TritonX100, 1% BSA, in 0.5M TRIS pH 8before use.

C) Preparation of Test Pads

A microfluidic device was built from stencil-cut laminates and containedmultiple detection chambers of the form illustrated in FIG. 9. Eachdetection chamber was formed with an inlet port and an outlet portfluidically connected to the detection chamber by microfluidic channels.Sufficient detection chambers were built for the experiment.

Before final assembly, test pads in the detection chamber were maskedand plasma treated with oxygen gas. Peptidyl hapten-specific antibodies(Research Diagnostics, Flanders N.J.) and negative control solution werespotted on the test pads, 1 uL per pad, and dried in place under vacuum.Each detection chamber contained one test pad corresponding to eachprimer set and a negative control. The fully assembled device wastreated with blocking/wash solution consisting of 0.9×PBS, 30 mg/mL BSAand 1% TritonX100 to passivate untreated plastic surfaces. The blockingsolution was removed before use and the chambers were dried.

D) Assay Protocol

Using known DNA samples from enteric pathogens, PCR was performed withthe prepared primer sets (above) for 35 cycles. Platinum QuantitativeRT-PCR Thermoscript One-Step System reagents were used for theamplification. Successful amplification was confirmed by 5% agarose gelelectrophoresis. Amplicon 10 uL was then resuspended with 5 uL of beads(above) in about 20 uL of buffer containing 10 mM MgCl₂, 0.5% BSA, 0.1%TritonX100 and 5 mM TRIS Buffer pH 8 and the bulk of this solution wasloaded into a detection chamber. Each amplicon product corresponded to asingle primer set and was loaded into a separate detection chamber.

The beads were first captured with a magnet positioned on the bottom ofthe detection chamber and the excess solution was removed. The magnetwas then used to smear the bead paste onto, through and across the testpads, and the mixture was then allowed to incubate 1 min. With themagnet positioned on the bottom of the well, the well was graduallyfilled with blocking solution. The magnet was moved along the flow ofthe buffer, creating a bead front on the bottom layer of the detectionchamber. The magnet was then shifted to the top of the detectionchamber, lifting unbound beads out of the test pad areas. The unboundmaterial could be resuspended in flowing buffer and rinsed to waste. Thetest pads were then rinsed with 1 volume of fresh buffer. Bright orangetest pad “stripes” were immediately visible and were determined tocorrectly reflect specificity of binding of the hapten-tagged ampliconto the test pad containing the complementary antibody. Because thedetection chambers were aligned in parallel when constructed, astairstep pattern was evident after all the amplicon bead mixtures wereprocessed because each tagged amplicon was bound by only one test pad ineach detection chamber.

Upon clearing, positive tests were immediately visible as bright orangebands corresponding to the location of particular test strips. Negativetest strips and negative controls remained translucent and uncolored.The results could be easily decoded by matching the location of thestained test pad with a key of the antibodies used in spotting.

EXAMPLE 2

PCR amplification was performed in a microfluidic device as follows:

A microfluidic device was built from stencil-cut laminates. Before finalassembly, biotin- and hapten-tagged primer pairs, dATP, dCTP, dGTP anddTTP, TAQ polymerase, and a matrix consisting of TritonX100, BSA, PEGand Trehalose plus magnesium chloride were deposited in theamplification channel or chamber and dried in place under vacuum.Streptavidin-coated magnetic beads (Dynal MyOne Streptavidin Cl,Carlsbad Calif.) were spotted and dried in a chamber adjoining theamplification channels or chambers. Test pad areas in the detectionchamber were stenciled (see FIG. 21 for general approach) and gas plasmatreated, before antibody solutions were applied and dried in place.Antibody spots were blocked with StabilCoat (SurModics, Eden PrairieMinn.). The device was then treated with a TritonX100:BSA buffer topassivate untreated plastic surfaces.

The following reagents were also prepared:

Lysis Buffer

4.5M Guanidinium thiocyanate

5% TritonX100

1% Sarcosine

50 mM MES, pH 5.5

20 mM EDTA

Wash Reagent

Anhydrous ethanol

Elution Buffer E11

1% TritonX100

0.1 mM EDTA

20 mM TRIS pH8.0

50 U RNAsin (Promega)

Rehydration Buffer

1% TritonX100

0.5% NaCl

10 mg/mL Bovine Serum Albumin

50 mM TRIS pH 8.0

Lysis Buffer, Wash Reagent, Elution Buffer, and Rehydration Buffer werealiquoted into sealed blister packs in designated chambers of thedevice. The device was then fully assembled and placed in a pneumaticcontroller with variable temperature TEC heating blocks positioned underthe PCR fluidics and thermal interface assembly.

Clinical swab samples from diarrhoeal patients known to containpathogenic microorganisms were handled with gloves in a biosafetycabinet. Each rectal swab was mixed vigorously with 400 uL of TE tosolubilize the contents. Using filter-plugged pipet tips, about 400 uLof homogenate was then transferred to the sample port of themicrofluidic device and the sample port was closed. All other steps wereperformed in the single-entry device, with no other operator exposure.

The remaining assay steps were automated.

An on-board sanitary bellows pump was used to pull sample through apre-filter consisting of a depth filter element, made of polypropylenefor example, supported on a laser-cut plastic ribs. A valve was thenused to close the sample port. The crude filtrate was then mixed withlysis buffer and drawn through a glass fiber filter to trap nucleicacids, and the filter retentate was rinsed thoroughly with ethanol. Allrinses were sequestered in an onboard waste receptacle which ventsthrough a 0.45 micron hydrophobic membrane filter. The nucleic acids onthe glass fiber membrane were then eluted with elution buffer and portedinto the reaction channel containing primers, dNTPs, polymerase,magnesium, buffer and surface active agents in dehydrated form. Thereaction mixture, in a volume of about 50 uL, was then heated to 95° C.in the PCR fluidics and thermal interface assembly for about 10 sec toeffect denaturation of double stranded sequences and secondary structurein the sample. Heating and cooling was supplied by external Peltierchips mounted on suitable heat sinks and PID controlled within a 1° C.range from setpoint. Immediately thereafter, the temperature wasreturned to about 60° C. for a first round of annealing and extension,which was continued for about 20 sec. Thermocycling was repeated for 40cycles over an 18 min period.

Following extraction and amplification, the amplicon products were movedto a mag mix chamber for mixing streptavidin-labelled magnetic beads(Dynal, MyOne Streptavidin C1) which had been rehydrated in RehydrationBuffer. This mixture was incubated with gentle mixing and thentransferred to a MagnaFlow chamber. Optionally the reaction mix can berinsed to remove unreacted hapten-conjugated primer while holding themagnetic beads in place. Using permanent magnets mounted on an X-Ystage, the coated beads with putative target amplicon were brought intocontact with the capture antibody test pads or array in the detectionchamber, and unbound beads were moved away from the test pads with amoving magnetic field and sent to waste. Primers and non-specificamplicons were rinsed from the chamber with an excess of rehydrationbuffer, which again was discarded into on-board waste.

Upon clearing, positive tests were readily visible as orange bandscorresponding to the location of particular test strips. Negative teststrips remained translucent and uncolored. Time following transfer ofamplification mixture to detection event was about 4 min. Knowing theidentity of each immobilized antibody, the results could be easilydecoded. In best practice to this date, the time from amplification todata presentation is less than 4 minutes.

In a test run with clinical samples, pathogens in 46 out of 47 stoolswere scored correctly in screening with the Magnaflow device. One samplepreviously identified as containing Salmonella by culture was identifiedas also containing enterotoxigenic E. Coli O157H1 by Magnaflow, (i.e., adouble infection). For this example, the following primer pairs wereobtained by custom synthesis and chemically conjugated by methods knownin the art.

5′-Peptidyl Oligomers 3′ Target Gene Primer Target Sequence Haptenconjugate InvA CAATGTAGAACGACCCCATAAACA EQKLISEEDL′ (SEQ ID NO:15) (SEQID NO:8) Gyrase A GCCATTCTAACCAAAGCATCATA DTYRYI′ (SEQ ID NO:16) (SEQ IDNO:13) ipaH ACTCCCGACACGCCATAGAA QPELAPEDPED′ (SEQ ID NO:17) (SEQ IDNO:14) Eae CTATCCAACAAGTTCAATTCATCC TDFYLK′ (SEQ ID NO:18) (SEQ IDNO:12) Stx1A AGACGTATGTAGATTCGCTGAA YTDIEMNRLGK′ (SEQ ID NO:19) (SEQ IDNO:10) Stx2A CTGGATGCATCTCTGGTCAT CEEEEYMPME′ (SEQ ID NO:20) (SEQ IDNO:11) Ma1B GGCGAATACCCAGCGACAT YPYDVPDYA′ (SEQ ID NO:21) (SEQ ID NO:9)5′-Biotinylated Oligomers 5′-Target Gene Primer Target Sequence PrimerConjugate InvA TATCTGGTTGATTTCCTGATCGC Biotin (SEQ ID NO:22) Gyrase AAAATGATGAGGCAAAAAGTAGAACA Biotin (SEQ ID NO:23) ipaH GGACATTGCCCGGGATAAABiotin (SEQ ID NO:24) Eae TTACCCGACGCCTCAAAC Biotin (SEQ ID NO:25) Stx1AAGACGTATGTAGATTCGCTGAA Biotin (SEQ ID NO:26) Stx2A GGAATGCAAATCAGTCGTCABiotin (SEQ ID NO:27) MalB GCCGATGCCAAATCGTCAG Biotin (SEQ ID NO:28)

Forward primers for this example were conjugated with biotin. Reverseprimers were conjugated with peptide haptens for which antibodies wereavailable (Research Diagnostics, Flanders N.J.). Covalent attachment ofthe haptens was at the 5′ terminus of the oligomer. Peptides wereactivated at the amino terminus for coupling.

EXAMPLE 3

A result of an assay in which the targets of Example 2 were extracted,amplified and detected is shown in FIG. 6.

EXAMPLE 4

A respiratory panel containing biotinylated and peptidyl hapten-taggedprimer pairs is designed. The primers are synthesized and then depositedin separate amplification channels or chambers of a device. Followingthe procedure of Example 2, throat swab washings are analyzed. Amini-bead impact mill is used to prepare the sample prior to analysis. Aresult is displayed in the detection chamber. The product is packaged asa kit.

EXAMPLE 5

A sexually transmitted disease panel containing biotinylated andpeptidyl hapten-tagged primer pairs is designed and the primers aresynthesized. The primers are then deposited in separate amplificationchannels or chambers of a device. Following the procedure of Example 2,vaginal swab washings are analyzed. A detection endpoint is displayed inthe detection chamber. The product is packaged as a kit.

EXAMPLE 6

An oncogene panel containing biotinylated and peptidyl hapten-taggedprimer pairs is designed and the primers are synthesized. The primersare then deposited in a common amplification channel or chamber.Following PCR amplification, the amplification products are detected ina detection station. The product is packaged as a kit.

While the above description contains specificities, these specificitiesshould not be construed as limitations on the scope of the invention,but rather as exemplifications of embodiments of the invention. That isto say, the foregoing description of the invention is exemplary forpurposes of illustration and explanation. Without departing from thespirit and scope of this invention, one skilled in the art can makevarious changes and modifications to the invention to adapt it tovarious usages and conditions without inventive step. As such, thesechanges and modifications are properly, equitably, and intended to bewithin the full range of equivalence of the following claims. Thus thescope of the invention should be determined by the appended claims andtheir legal equivalents, rather than by the examples given.

1-4. (canceled)
 5. A method for multiplex target nucleic acid detectionby heterogeneous binding assay, comprising: a) preparing anamplification reagent comprising an amplification primer set having afirst primer tagged with a peptidyl hapten tag and a second primertagged with a ligand tag, wherein said first primer and said secondprimer are complementary to flanking sequences of a nucleic acid targetsequence to be assayed; b) preparing a paramagnetic bead reagentcomprising a paramagnetic microbead with a binding agent, wherein saidbinding agent has a binding affinity for said ligand tag of said secondprimer; c) preparing a test pad area with immobilized capture antibody,wherein said capture antibody has a binding affinity for said peptidylhapten tag of said first primer; d) processing a biological sample torelease a nucleic acid fraction; e) contacting said nucleic acidfraction with said amplification reagent; f) performing an amplificationto yield amplification products; and g) assaying said amplificationproducts for a two-tailed amplicon labeled with said peptidyl hapten tagof said first primer on a first end of said two-tailed amplicon and saidligand tag of said second primer on a second end of said two-tailedamplicon by: i) contacting said amplification products with saidparamagnetic bead reagent, thereby binding said ligand tag of saidsecond end of said two-tailed amplicon to said binding agent of saidparamagnetic bead reagent to yield a two-tailed amplicon paramagneticbead complex; ii) sweeping said two-tailed amplicon paramagnetic beadcomplex into close contact with said test pad area by moving a magneticforce field from outside to inside said test pad area, thereby bindingsaid peptidyl hapten tag of said first end of said two-tailed ampliconof said two-tailed amplicon paramagnetic bead complex to said captureantibody of said test pad area to yield an immunoimmobilizedparamagnetic reporter complex; iii) sweeping from said test pad area anyunbound two-tailed amplicon paramagnetic bead complex by moving saidmagnetic force field from inside to outside said test pad area; and iv)detecting the presence of said immunoimmobilized paramagnetic reportercomplex on said test pad area.
 6. The method of claim 5 wherein saidligand tag of said amplification reagent is biotin and said bindingagent of said paramagnetic bead reagent is an avidin.
 7. The method ofclaim 5 wherein said peptidyl hapten tag is a peptide with 2 to 100amino acid residues.
 8. The method of claim 5 wherein saidimmunoimmobilized paramagnetic complex on said test pad is detectedvisually.
 9. The method of claim 5 wherein said amplification stepcomprises a thermocycling protocol or isothermal protocol.
 10. Themethod of claim 5 wherein said amplification step further comprisesreverse transcription.
 11. The method of claim 5 wherein saidamplification step comprises nested amplification.
 12. The method ofclaim 5 further comprising a multiplex target detection protocol.
 13. Akit for performing the method of claim 5, said kit comprising saidamplification reagent and said test pad area. 14-33. (canceled)
 34. Amolecular detection complex comprising a two-tailed amplicon having afirst end and a second end, said first end comprising a first primercovalently conjugated with a peptidyl hapten tag, and said second endcomprising a second primer covalently conjugated with a ligand tag,wherein said first end further comprises a peptidyl hapten-boundanti-peptidyl hapten antibody immobilized on a solid phase, and whereinsaid second end further comprises a ligand-bound ligand bindingagent-coated reporter group.
 35. The molecular detection complex ofclaim 34, wherein said ligand-bound ligand binding agent-coated reportergroup is a magnetic microbead coated with said ligand binding agent, andwherein said solid phase is a test pad area.
 36. An apparatus forforming and purifying the molecular detection complex of claim 35,comprising: a) a microfluidic cartridge comprising a substrate and amicrochannel in said substrate, said microchannel comprising a fluidpath with axis of flow and with upper and lower aspects; b) a test padin said microchannel, said test pad comprising an affinity capture agentcomprising an anti-peptidyl hapten antibody immobilized on a solidphase; c) a means for introducing a fluid comprising (i) a paramagneticmicrobead comprising said reporter group and (ii) a two-tailed ampliconinto said microchannel; and d) a means for moving a magnetic force fieldalong a plane parallel to said axis of flow of said microfluidic channelto (i) sweep said paramagnetic microbead in said fluid into closecontact with said affinity capture agent, thereby binding saidparamagnetic microbead to said affinity capture agent and binding saidtwo-tailed amplicon to said paramagnetic microbead to yield saidmolecular detection complex, and (ii) sweep from said test pad anyunbound paramagnetic microbead and any unbound two-tailed amplicon.37-43. (canceled)
 44. A method for rapid bioassay comprising: a)preparing an amplification primer set comprising a first primercomprising a peptidyl hapten tag and a second primer comprising a ligandtag; b) forming a two-tailed amplicon product comprising said peptidylhapten tag of said first primer on a first end and said ligand tag ofsaid second primer on a second end by amplifying a nucleic acid targetwith said amplification primer set; c) complexing said two-tailedamplicon product to a reporter group having a binding affinity for saidligand tag to yield a two-tailed amplicon reporter group complex; d)capturing said two-tailed amplicon reporter group complex on a solidphase having an immobilized capture antibody, wherein said captureantibody has a binding affinity for said peptidyl hapten tag, to yieldan immobilized reporter group complex; and, e) detecting saidimmobilized reporter group complex.
 45. The method of claim 44 whereinsaid reporter group is a magnetic microbead and said solid phase is atest pad.
 46. The method of claim 44 wherein said reporter group is afluorophore and said solid phase is a barcoded latex bead.
 47. Themethod of claim 44, wherein said steps a) through c) are performed for aplurality of first primers conjugated with different peptidyl haptentags and a plurality of two-tailed amplicon products are captured anddetected on a plurality of solid phases, each with an antibody specificfor one of said different peptidyl hapten tags.
 48. The method of claim45, wherein said steps a) through c) are performed for a plurality offirst primers conjugated with different peptidyl hapten tags and aplurality of two-tailed amplicon products are magnetically captured anddetected on a plurality of solid test pads, each with an antibodyspecific for one of said different peptidyl hapten tags.
 49. The methodfor multiplex target nucleic acid detection by heterogeneous bindingassay of claim 12, wherein said amplification reagent comprises aplurality of first primers, each tagged with a different peptidyl haptentag, and wherein said test pad area comprises a plurality of test pads,each with an immobilized monoclonal capture antibody having a bindingaffinity for one of the peptidyl hapten tags of the plurality of firstprimers.
 50. The method for multiplex target nucleic acid detection byheterogeneous binding assay of claim 12, wherein said test pad areacomprises a plurality of test pads, each with an immobilized monoclonalcapture antibody having a binding affinity for a different peptidylhapten tagged amplicon.
 51. A method for multiplex target nucleic aciddetection by heterogeneous binding assay, comprising: a) preparing aplurality of amplification reagents, each comprising an amplificationprimer set having a first primer tagged with a peptidyl hapten tag and asecond primer tagged with an affinity tag, wherein each pair of saidfirst primers and said second primers are complementary to flankingsequences of a plurality of nucleic acid target sequences to be assayed;b) preparing a plurality of paramagnetic bead reagents, each comprisinga paramagnetic microbead with a binding agent, wherein each of saidbinding agents has a binding affinity for one of said affinity tags ofsaid second primers; c) preparing a plurality of test pads in a test padarea, each test pad having a different immobilized capture antibodyhaving a binding affinity for one of said peptidyl hapten tags of saidfirst primers; d) processing a biological sample to release a nucleicacid fraction; e) contacting said nucleic acid fraction with saidamplification reagents; f) performing an amplification to yieldamplification products; and g) assaying said amplification products fortwo-tailed amplicons labeled with one of said peptidyl hapten tags ofsaid first primers on a first end and one of said affinity tags of saidsecond primers on a second end by: i) contacting said amplificationproducts with said paramagnetic bead reagents, thereby binding saidaffinity tags of said second ends of said two-tailed amplicons to saidbinding agents of said paramagnetic bead reagents to yield two-tailedamplicon paramagnetic bead complexes; ii) sweeping said two-tailedamplicon paramagnetic bead complexes into close contact with saidplurality of test pads by moving a magnetic force field from outside toinside said test pad area, thereby binding said peptidyl hapten tags ofsaid first ends of said two-tailed amplicon of said two-tailed ampliconparamagnetic bead complex to said capture antibodies of said test padsto yield immobilized paramagnetic reporter complexes; iii) sweeping fromsaid test pad area any unbound two-tailed amplicon paramagnetic beadcomplexes by moving said magnetic force field from inside to outsidesaid test pad area; and iv) detecting the presence of saidimmunoimmobilized paramagnetic reporter complexes on each of said testpads.
 52. The method for multiplex target nucleic acid detection byheterogeneous binding assay of claim 51, wherein each of said affinitytags is a peptidyl hapten tag and each of said binding agents is ananti-peptidyl hapten antibody.
 53. A molecular detection complexcomprising a two-tailed amplicon having a first end and a second end,said first end comprising a first primer covalently conjugated with afirst peptidyl hapten tag, and said second end comprising a secondprimer covalently conjugated with a second peptidyl hapten tag, whereinsaid first end further comprises a first peptidyl hapten-boundanti-first peptidyl hapten antibody immobilized on a solid phase, andwherein said second end further comprises a second peptidyl hapten-boundanti-second peptidyl hapten antibody-coated reporter group.
 54. A methodfor rapid bioassay comprising: a) preparing an amplification primer setcomprising a first primer comprising a first peptidyl hapten tag and asecond primer comprising a second peptidyl hapten tag; b) forming atwo-tailed amplicon product comprising said first peptidyl hapten tag ofsaid first primer on a first end and said second peptidyl hapten tag ofsaid second primer on a second end by amplifying a nucleic acid targetwith said amplification primer set; c) complexing said two-tailedamplicon product to a reporter group having a binding affinity for saidsecond peptidyl hapten tag of said second primer to yield a two-tailedamplicon reporter group complex; d) capturing said two-tailed ampliconreporter group complex on a solid phase having an immobilized captureantibody, wherein said capture antibody has a binding affinity for saidfirst peptidyl hapten tag, to yield an immobilized reporter groupcomplex; and e) detecting said immobilized reporter group complex.
 55. Akit for performing a multiplex nucleic acid bioassay with multiplexdetection of two-tailed amplicons, said kit comprising: a first assayreagent having a peptidyl-hapten conjugated first amplification primer;a plurality of test pads, each of said test pad comprising a dehydratedfirst affinity binding agent with affinity for said peptidyl-hapten ofsaid first primer; and a second assay reagent comprising a bead reagentas a reporter group, wherein said bead reagent is coated with a secondaffinity binding agent.
 56. The kit of claim 55 for performing a panelassay for multiplex detection of multiple nucleic acid targets.